Formulations of dengue virus vaccine compositions

ABSTRACT

The present invention relates to formulations of dengue virus vaccine comprising at least one live attenuated dengue virus or live attenuated chimeric flavivirus, a buffer, a sugar, a cellulose derivative, a glycol or sugar alcohol, optionally an alkali or alkaline salt and an amino acid; and formulations of dengue virus vaccine comprising at least one live attenuated dengue virus or live attenuated chimeric flavivirus, a buffer, a sugar of at least 150 mg/ml, a carrier, and optionally an alkali or alkaline salt and an amino acid.

FIELD OF THE INVENTION

The present invention relates to formulations of dengue virus vaccinecomprising at least one live, attenuated dengue virus or live,attenuated chimeric flavivirus, a buffer, a sugar, a cellulosederivative and a sugar alcohol or glycol, and optionally an amino acidand an alkali or alkaline salt; and formulations of dengue virus vaccinecomprising at least one live, attenuated dengue virus or live,attenuated chimeric flavivirus, a buffer, a sugar of at least 150 mg/ml,a carrier, and optionally an alkali or alkaline salt, or, alkali oralkaline salt and an amino acid.

BACKGROUND OF THE INVENTION

The family Flaviviridae includes the prototype yellow fever virus (YF),the four serotypes of dengue virus (DENV-1, DENV-2, DENV-3, and DENV-4),Japanese encephalitis virus (JE), tick-borne encephalitis virus (TBE),West Nile virus (WN), Saint Louis encephalitis virus (SLE), and about 70other disease causing viruses. Flaviviruses are small, enveloped virusescontaining a single, positive-strand RNA genome. Ten gene products areencoded by a single open reading frame and are translated as apolyprotein organized in the order: capsid (C), “preMembrane” (prM,which is processed to “Membrane” (M) just prior to virion release fromthe cell), “envelope” (E), followed by non-structural (NS) proteins NS1,NS2a, NS2b, NS3, NS4a, NS4b and NS5 (reviewed in Chambers, T. J. et al.,Annual Rev Microbiol (1990) 44:649-688; Henchal, E. A. and Putnak, J.R., Clin Microbiol Rev. (1990) 3:376-396). Individual flaviviralproteins are then produced through precise processing events mediated bythe host as well as virally encoded proteases.

The envelope of flaviviruses is derived from the host cell membrane andcontains the virally-encoded membrane anchored membrane (M) and envelope(E) glycoproteins. The E glycoprotein is the largest viral structuralprotein and contains functional domains responsible for cell surfaceattachment and intra-endosomal fusion activities. It is also a majortarget of the host immune system, inducing the production of virusneutralizing antibodies, which are associated with protective immunity.

Dengue viruses are transmitted to man by mosquitoes of the genus Aedes,primarily A. aegypti and A. albopictus. Infection by dengue virusesleads to a diverse clinical picture ranging from an inapparent or mildfebrile illness, through classical dengue fever (DF), to denguehemorrhagic fever/dengue shock syndrome (DHF/DSS). Dengue fever ischaracterized by high fever, headache, joint and muscle pain, rash,lymphadenopathy and leucopenia (Gibbons, R. V. and D. W. Vaughn, BritishMedical Journal (2002) 324:1563-1566). DHF/DSS is a more severe form ofinfection more common in children, marked by vascular permeabilityand/or severe hemorrhagic manifestations ranging from the presence ofpetechiae and ecchymosis to spontaneous severe hemorrhage and profoundshock. Without diagnosis and prompt medical intervention, the suddenonset and rapid progression of DHF/DSS can be fatal if untreated.

Dengue viruses are the most significant group of arthropod-transmittedviruses in terms of global morbidity and mortality with an estimated onehundred million dengue infections occurring annually including at least36 million cases of dengue fever and 250,000 to 500,000 cases of DHF/DSS(Gubler, D. J., Clin. Microbiol. Rev. (1998) 11:480-496; Gibbons,supra). With the global increase in population, urbanization of thepopulation especially throughout the tropics, and the lack of sustainedmosquito control measures, the mosquito vectors of dengue have expandedtheir distribution throughout the tropics, subtropics, and sometemperate areas, bringing the risk of dengue infection to over half theworld's population. Modern jet travel and human emigration havefacilitated global distribution of dengue serotypes, such that multipleserotypes of dengue are now endemic in many regions. There has been anincrease in the frequency of dengue epidemics and the incidence ofDHF/DSS in the last 20 or more years. For example, in Southeast Asia,DHF/DSS is a leading cause of hospitalization and death among children(Gubler, supra; Gibbons and Vaughn, supra).

To date, the development of flavivirus vaccines has been met with mixedsuccess. There are four basic approaches that have been implemented inan effort to produce vaccine candidates to protect against diseasecaused by flaviviruses: live-attenuated, inactivated whole virus,recombinant subunit protein, and DNA-based vaccines. A live-attenuatedvaccine for yellow fever virus has been available for decades and morerecently a live attenuated vaccine for Japanese encephalitis has beenregistered in various countries around the world. The use of inactivatedwhole virus vaccines has been demonstrated for TBE and JE viruses withseveral registered products available. Heinz et al. Flavivirus andflavivirus vaccines. Vaccine 30: 4301-06 (2012).

Despite the successes of the YF, JE, and TBE vaccines highlighted above,the use of live-attenuated virus and inactivated virus methods todevelop vaccines for dengue virus has been met with significantchallenges. There are four serotypes of dengue virus (DENV1, DENV2,DENV3, and DENV4) and strains of each serotype are found circulatingthroughout the dengue endemic regions of the world. Natural infectionconfers long lasting immunity to the infecting serotype but not to otherdengue serotypes. The more severe forms of the disease (DHF/DSS) occurmost often after secondary dengue infection, when infection with oneserotype of dengue virus is followed by a second infection with anotherserotype. The more frequent association of DHF and DSS with secondarydengue infection has been hypothesized to be due to non-neutralizingantibodies induced by infection with one virus type enhancinginfectivity of a second dengue virus type (antibody-dependentenhancement—ADE).

To date, the majority of the vaccines tested clinically are live,attenuated vaccines. The use of non-replicating vaccine candidates isalso being explored. For example, Ivy et al. (U.S. Pat. No. 6,432,411)disclose a tetravalent subunit vaccine comprising DEN1-4 80% E (thepeptide region of DEN1-4 corresponding to amino acids 1-395 of theDENV-2 envelope polypeptide) proteins. Ivy et al, supra, also reportcompositions comprising DENV 1-4 80% E and ISCOMATRIX® adjuvant. Colleret al. (WO 2012/154202) disclose tetravalent formulations comprisingDEN1-4 80% E of DEN 1-4. Inactivated viruses may also be used aspotential vaccine candidates or as components of an effective vaccine(Putnak et al. Vaccine 23: 4442-4452 (2005), U.S. Pat. Nos. 6,190,859,6,254,873 and Sterner et al. WO 2007/002470). Compositions comprising alive attenuated dengue virus vaccine and a non-replicating denguevaccine are disclosed in International Patent Application No.PCT/US14/042625 (WO2014/204892).

Whole viruses are one of the commonly used antigens in several vaccineproducts due to their ability to generate humoral and cellular immuneresponses. Vaccine products containing whole viruses are challenging tostabilize as these are sensitive to heat, freeze/thaw and otherprocessing stresses leading to significant potency losses. Theseproducts are typically stored frozen (below −20° C.) or as dried powder.Frozen products are not easy to store and distribute as they need astringent cold-chain requirement to prevent potency loss. Drying ofwhole viruses, especially enveloped viruses, often leads to significantloss of potency due to the freezing and drying stresses encounteredduring the drying process. Therefore, there is a need in the art togenerate stable formulations of Dengue virus.

SUMMARY OF THE INVENTION

The current invention provides stable formulations of live attenuateddengue vaccine. The addition of a cellulose derivative and a sugaralcohol or glycol improved stability and/or yield after drying.Alternatively, the addition of sugar of at least 150 mg/ml improvedstability and/or yield after microwave drying.

In one aspect, the invention provides a formulation comprising a liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus (LACV),a buffer at pH about 6.5 to 8.5, a sugar, a glycol or sugar alcohol, anda cellulose derivative selected from the group consisting ofcarboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC),crosscarmellose, and methyl cellulose, or a pharmaceutically acceptablesalt thereof optionally an alkali or alkaline salt, and optionally anamino acid selected from the group consisting of Ala, Asp, His, Leu,Lys, Gln, Pro or Glu, or a combination thereof.

In one embodiment, the buffer is selected from the group consisting ofsuccinate, histidine, phosphate, TRIS, Bis-Tris, MES, MOPS, HEPES,acetate and citrate, or a combination thereof. In another embodiment,the alkali or alkaline salt is magnesium chloride, calcium chloride,potassium chloride, sodium chloride or a combination thereof. In afurther embodiment, the sugar is trehalose or sucrose. In oneembodiment, the cellulose derivative is a pharmaceutically acceptablesalt of carboxymethyl cellulose. In another embodiment, the glycol isselected from the group consisting of propylene glycol, polypropyleneglycol, ethylene glycol, polyethylene glycol, and polyethylene glycolmonomethyl ethers. In a further embodiment, the sugar alcohol isglycerol.

In another aspect, the formulation comprises a live attenuated denguevaccine comprising at least one live attenuated dengue virus (LAV) or atleast one live attenuated chimeric flavivirus at about 100-10,000,000pfu/ml, a buffer at pH about 6.5 to 8.5, about 50-300 mg/ml sugar, about2.5-10.0 mg/ml propylene glycol (PG) or glycerol, and about 0.3-10 mg/mlsodium carboxymethylcellulose (sodium CMC), optionally about 10-150 mMNaCl, and optionally about 10-100 mM amino acid selected from the groupconsisting of Ala, Asp, His, Leu, Lys, Gln, Pro or Glu, or a combinationthereof a live attenuated dengue vaccine at about 100-100,000 pfu/ml,about 5-300 mM histidine, TRIS, Bis-Tris or phosphate buffer, or acombination thereof at pH about 7.0 to 8.0, about 50-300 mg/ml sugar,about 3-10 mg/ml propylene glycol or glycerol, and about 3-10 mg/mlsodium carboxymethylcellulose, optionally about 15-75 mM NaCl, andoptionally about 10-75 mM amino acid selected from the group consistingof Ala, Asp, His, Leu, Lys, Gln, Pro or Glu, or a combination thereof alive attenuated dengue vaccine at about 600-20,000 pfu/ml, about 5-300mM potassium phosphate buffer at pH about 7.0-8.0, about 60-120 mg/mlsucrose or trehalose or a combination thereof, about 3-7 mg/ml propyleneglycol or glycerol, and about 3-7 mg/ml sodium carboxymethylcellulosewith average molecular weight of about 90,000, and about 30-90 mM NaCl,and optionally about 10-75 mM amino acid Leu, Lys or Glu, or acombination thereof; a live attenuated dengue vaccine at about600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pH about7.0-8.0, about 90 mg/ml sucrose, about 5 mg/ml propylene glycol orglycerol, about 5 mg/ml sodium carboxymethylcellulose with averagemolecular weight of about 90,000, and about 75 mM NaCl; a liveattenuated dengue vaccine at about 600-20,000 pfu/ml, about 11 mMpotassium phosphate buffer at pH about 7.0-8.0, about 90 mg/ml sucrose,about 5 mg/ml propylene glycol, about 5 mg/ml sodiumcarboxymethylcellulose with average molecular weight of about 90,000,about 50 mM NaCl, and about 25 mM Leu; or a live attenuated denguevaccine at about 600-20,000 pfu/ml, about 11 mM potassium phosphatebuffer at pH about 7.5, about 90 mg/ml sucrose, about 5 mg/ml propyleneglycol, about 5 mg/ml sodium carboxymethylcellulose with averagemolecular weight of about 90,000, and about 30 mM NaCl. In one aspect ofthe foregoing embodiments, the formulation further comprises about90-200 mg/ml trehalose.

In a preferred embodiment of the invention, the formulation comprises alive attenuated dengue vaccine comprising at least one live attenuateddengue virus (LAV) or at least one live attenuated chimeric flavivirusat about 600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pHabout 7.5-8, about 90 mg/ml sucrose, about 110 mg/ml trehalose, about 5mg/ml propylene glycol, about 5 mg/ml sodium carboxymethylcellulose withaverage molecular weight of about 90,000, about 50 mM NaCl, and about 25mM Leu. In one aspect of the foregoing embodiments, the formulationfurther comprises a surfactant selected from poloxamer 188 and poloxamer407 at about 0.0001 to 5% w/v.

The invention also provides a formulation that comprises a liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus at about100-10,000,000 pfu/ml, a buffer at pH about 6.5 to 8.5, a sugar at about150-300 mg/ml, a carrier selected from the group consisting ofpolyvinylpyrrolidone (PVP), carboxymethyl cellulose, hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethylcellulose (2-HEC), crosscarmellose, methyl cellulose or apharmaceutically acceptable salt thereof, Human Serum Albumin (HSA) andgelatin; optionally an alkali salt or alkaline salt at about 5-100 mM;and optionally an amino acid Gln, Pro or Glu, or a combination thereof.

In one embodiment, the buffer is selected from the group consisting ofsuccinate, histidine, phosphate, TRIS, Bis-Tris, MES, MOPS, HEPES,acetate and citrate, or a combination thereof. In another embodiment,the alkali or alkaline salt is magnesium chloride, calcium chloride,potassium chloride, sodium chloride or a combination thereof. In afurther embodiment, the sugar is trehalose or sucrose, or a combinationthereof. In one embodiment, the sucrose to trehalose ratio is between1:1 to 1:4. In another embodiment, the carrier is a sodium carboxymethylcellulose, HPMC, HSA or gelatin.

In a further aspect, the invention provides formulations of a liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus at about200-100,000 pfu/ml, a buffer at pH about 6.5-8.0, about 150-300 mg/mlsugar as a combination of sucrose and trehalose, about 0.3 to 40 mg/mlsodium CMC, HSA, HPMC or gelatin, optionally about 10-100 mM alkali oralkaline salt, and optionally about 5-25 mM glutamic acid; a liveattenuated dengue vaccine at about 600-20,000 pfu/ml, about 5-300 mMhistidine, TRIS or phosphate buffer, or a combination thereof at pHabout 7.0 to 8.0, about 50-100 mg/ml sucrose, about 90-200 mg/mltrehalose, about 0.3-10 mg/ml sodium CMC or about 10-40 mg/ml gelatin,and about 30-90 mM alkali or alkaline salt; a live attenuated denguevaccine at about 600-20,000 pfu/ml, about 5-20 mM potassium phosphate atpH about 7-8, about 75 mg/ml sucrose, about 175 mg/ml trehalose, about 5mg/ml sodium CMC with average molecular weight of about 90,000, andabout 30 mM NaCl; a live attenuated dengue vaccine at about 600-20,000pfu/ml, about 5-20 mM potassium phosphate at pH about 7.0-8.0, about 75mg/ml sucrose, about 175 mg/ml trehalose, about 25 mg/ml gelatin, andabout 30 mM NaCl; or a live attenuated dengue vaccine at about600-20,000 pfu/ml, about 5-20 mM potassium phosphate at pH about7.0-8.0, about 250 mg/ml sucrose, and about 50 mg/ml PVP K12. In oneaspect of the foregoing embodiments, the formulation further comprises asurfactant selected from poloxamer 188 and poloxamer 407 at about 0.0001to 5% w/v.

In certain aspects of the foregoing embodiments, the formulation furthercomprises an aluminum adjuvant. The above formulations can be frozen orlyophilized, or reconstituted in solution. In one embodiment, thereconstitution is performed with about 0.5-1.0 ml saline solution, wateror Bacteriostatic Water for Injection (BWFI) and optionally a diluentcomprising an aluminum adjuvant. In another embodiment, the formulationis the aqueous solution prior to lyophilization or microwave vacuumdrying.

In one embodiment, the live attenuated dengue vaccine comprisestetravalent live attenuated dengue virus or live attenuated chimericflavivirus. In another embodiment, the LAV or the LACV comprise a viralgenome that contains a deletion of about 30 nucleotides corresponding tothe TL-2 stem-loop structure of the 3′ untranslated (UTR) region; whichreduces the replicative capacity of the virus. In a further embodiment,the live attenuated dengue virus is an LAV that comprise a viral genomethat contains a deletion of about 30 nucleotides corresponding to theTL-2 stem-loop structure of the 3′ untranslated (UTR) region, and isimmunogenic against dengue serotype 3, wherein the viral genome of theLAV further contains a deletion of nucleotides upstream from the Δ30deletion corresponding to the TL-3 structure of the 3′UTR.

In preferred embodiments of the invention, the live attenuated denguevaccine is a live attenuated tetravalent vaccine comprising a DEN1Δ30virus, a DEN2/4Δ30 virus (a DEN2 Δ30LACV on a DEN4 backbone), a DEN3Δ30virus and a DEN4Δ30 virus. In another preferred embodiment, the liveattenuated dengue virus is an LAV comprising rDEN1Δ30-1545, rDEN2/4Δ30(ME)-1495,7163, rDEN3Δ30/31-7164, and rDEN4Δ30-7132,7163,8308.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effect of Sodium CMC, PG, amino acids on DENV4 lyophilizationyield for DEN4 formulations.

FIG. 2: Effect of Sodium CMC, PG, amino acids on DENV4 stability forDEN4 formulations. Formulation 26 (*) was not tested due to cakecollapse after storage at 25° C.

FIG. 3: Effect of sugar alcohol on DENV4 lyophilization yield for DEN4formulations.

FIG. 4: Effect of sugar alcohol on DENV4 stability for DEN4formulations.

FIG. 5: Effect of pH on DENV4 lyophilization yield for DEN4formulations.

FIG. 6: Effect of pH on DENV4 stability for DEN4 formulations.

FIG. 7: Effect of buffer on DENV4 lyophilization yield for DEN4formulations.

FIG. 8: Effect of buffer on DENV4 stability for DEN4 formulations.

FIG. 9: Effect of NaCl concentration on DENV4 lyophilization yield. forDEN4 formulations.

FIG. 10: Effect of NaCl concentration on DENV4 stability for DEN4formulations.

FIG. 11: Effect of propylene glycol and glyercol on lyophilizationyields of Dengue serotypes.

FIG. 12: Effect of propylene glycol and glycerol on stability of Dengueserotypes.

FIG. 13: Effect of L-15 concentration on relative potency for frozen,microwave dried (MVD) and lyophilized (lyo) DEN1 formulations.

FIG. 14: Effect of L-15 concentration on relative potency for frozen,microwave dried (MVD) and lyophilized (lyo) DEN2 formulations.

FIG. 15: Effect of L-15 concentration on relative potency for frozen,microwave dried (MVD) and lyophilized (lyo) DEN3 formulations.

FIG. 16: Effect of L-15 concentration on relative potency for frozen,microwave dried (MVD) and lyophilized (lyo) DEN4 formulations.

FIG. 17: Relative potency for frozen, microwave dried (MVD) andlyophilized (lyo) DEN1 formulations.

FIG. 18A-B: A) Stability of tetravalent formulations at 37° C. after oneweek. B) Stability of tetravalent formulations at 25° C. after onemonth.

FIG. 19A-D: Stability of tetravalent formulations (DEN1-DEN4) at 2-8° C.tested every 3 months up to 18 months.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

Reference to “or” indicates either or both possibilities unless thecontext clearly dictates one of the indicated possibilities. In somecases, “and/or” was employed to highlight either or both possibilities.

The term “about”, when modifying the quantity (e.g., mM, or M) of asubstance or composition, the percentage (v/v or w/v) of a formulationcomponent, the pH of a solution/formulation, or the value of a parametercharacterizing a step in a method, or the like refers to variation inthe numerical quantity that can occur, for example, through typicalmeasuring, handling and sampling procedures involved in the preparation,characterization and/or use of the substance or composition; throughinstrumental error in these procedures; through differences in themanufacture, source, or purity of the ingredients employed to make oruse the compositions or carry out the procedures; and the like. Incertain embodiments, “about” can mean a variation of ±0.1%, 0.5%, 1%,2%, 3%, 4%, 5%, or 10%.

The term “bulking agents” comprise agents that provide the structure ofthe freeze-dried product. Common examples used for bulking agentsinclude mannitol, glycine, and lactose. In addition to providing apharmaceutically elegant cake, bulking agents may also impart usefulqualities in regard to modifying the collapse temperature, providingfreeze-thaw protection, and enhancing the protein stability overlong-term storage. These agents can also serve as tonicity modifiers.

The “Dengue Virus reference sample” has the same dengue virusformulation components and ratios as the dengue virus formulation testsample, and refers to the solid composition immediately after drying thedengue virus formulation under the same conditions as the dengue virusformulation test sample (i.e. lyophilization, microwave dried, lyospheredried), or the foregoing dried solid composition stored at conditionswhere there is no or minimal infectivity loss of the dengue virus (i.e.stored at or below −70° C.); or the frozen solid dengue virusformulation at −70° C.

“Glycol” refers to a chemical compound with two hydroxyl groups.

“Infectivity loss” refers to comparing the loss of viral replication ofa dengue virus test sample to a dengue virus reference sample usingmethods known in the art. In one embodiment, the infectivity loss ismeasured using a dengue relative infectivity assay. In anotherembodiment, the infectivity loss is measured using a plaque assay.

The terms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.An excipient may be included in pre-lyophilized formulations to enhancestability of the lyophilized product upon storage.

“Lyosphere,” as used herein, refers to dried frozen unitary bodiescomprising a therapeutically active agent which are substantiallyspherical or ovoid-shape. In some embodiments, the lyosphere diameter isfrom about 2 to about 12 mm, preferably from 2 to 8 mm, such as from 2.5to 6 mm or 2.5 to 5 mm. In some embodiments, the volume of the lyosphereis from about 20 to 550 μL, preferably from 20 to 100 μL, such as from20 to 50 μL. In embodiments wherein the lyosphere is not substantiallyspherical, the size of the lyosphere can be described with respect toits aspect ratio, which is the ratio of the longer dimension to theshorter dimension. The aspect ratio of the lyospheres can be from 0.5 to2.5, preferably from 0.75 to 2, such as from 1 to 1.5.

“Microwave Vacuum Drying” as used herein, refers to a drying method thatutilizes microwave radiation (also known as radiant energy ornon-ionizing radiation) for the formation of dried vaccine products(preferably, <6% moisture) of a vaccine formulation through sublimation.In certain embodiments, the microwave drying is performed as describedin US2016/0228532. In one embodiment, the microwave radiation is intraveling wave format.

A “reconstituted solution”, as used herein, is one that has beenprepared by dissolving dried virus in solid form (such as a lyophilizedcake) in a diluent such that the virus is dispersed in the reconstitutedsolution. The reconstituted solution is suitable for administration,(e.g. intramuscular administration), and may optionally be suitable forsubcutaneous administration.

“Salt(s)”, as employed herein, denotes acidic salts formed withinorganic and/or organic acids, as well as basic salts formed withinorganic and/or organic bases.

Pharmaceutically acceptable (i.e., non-toxic, physiologicallyacceptable) salts are preferred, although other salts are also useful.Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, zinc salts, salts with organic bases (forexample, organic amines) such as N-Me-D-glucamine, Choline,tromethamine, dicyclohexylamines, t-butyl amines, and salts with aminoacids such as arginine, lysine and the like.

“Sugar alcohol” refers to polyols derived from a sugar and have thegeneral formula HOCH₂(CHOH)_(n)CH₂OH, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Examples include but are not limited to mannitol, sorbitol, erythritol,xylitol and glycerol.

As used herein, “x % (w/v)” is equivalent to x g/100 ml (for example 5%w/v equals 50 mg/ml).

The term “live attenuated dengue virus,” also referred to as “LAV”herein, means the ability of the dengue virus to cause disease isreduced compared to wild-type dengue virus. One skilled in the art wouldunderstand that viruses may undergo mutation when cultured, passaged orpropagated. The LAV may contain these naturally occurring mutations, inaddition to mutations introduced for cloning purposes. The LAV may be ahomogenous or heterogeneous population with none, or one or more ofthese mutations.

The term “live attenuated chimeric virus” (alternatively “liveattenuated chimeric flavivirus”) or “LACV” refers to a live attenuatedchimeric virus wherein the viral genome comprises a backbone of a firstflavivirus (including C, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 genes)and the preMembrane (prM) and envelope (E) genes of a second flavivirus,wherein the second flavivirus is selected from DENV1, DENV2, DENV3 orDENV4. The first flavivirus can be a different dengue serotype oranother flavivirus, such as yellow fever virus.

The term “Δ30 LAV” refers to a live attenuated DEN1, DEN2, DEN3, or DEN4virus, wherein the LAV comprises a viral genome that contains a deletionof about 30 nucleotides (nt) corresponding to the TL2 stem-loopstructure of the 3′ untranslated (UTR) region from about nt 143 to aboutnt 172, which reduces the replicative capacity of the virus (see WO03/092592 and Whitehead et al., U.S. Pat. No. 8,337,860).

The term “Δ30 LACV” refers to a live attenuated chimeric flavivirus(LACV) from DENV 1-4 wherein the LACV comprises a viral genome thatcontains a deletion of about 30 nt corresponding to the TL2 stem-loopstructure of the 3′ UTR region from about nt 143 to about nt 172, whichreduces the replicative capacity of the virus (see WO 03/092592 andWhitehead et al., U.S. Pat. No. 8,337,860).

The term “Δ30/Δ31 LAV” refers to a live attenuated DEN1, DEN2, DEN3, orDEN4 virus, wherein the viral genome comprises a deletion of about 30 ntof the TL2 stem-loop structure of the 3′ UTR, and further comprises aseparate, noncontiguous, upstream deletion of about 31 nt at about nt258-228 of the 3′ UTR which removes sequence up to and including theTL-3 homologous structure so that the deletion extends as far as the 5′boundary of the TL-3 homologous structure of the dengue 3′UTR. SeeWhitehead et al., U.S. Pat. No. 8,337,860. In preferred embodiments ofthe invention, the DEN3 LAV comprises the Δ30/Δ31 mutations.

The term “Δ30/Δ31 LACV” refers to a live attenuated chimeric DEN1, DEN2,DEN3, or DEN4 virus as described above, wherein the viral genome of thechimeric virus comprises a 30 nt deletion of the TL2 stem-loop structureof the 3′ UTR, and further comprises a separate, noncontiguous, upstream31 nt deletion of the 3′ UTR, which deletes the TL-3 structure, asdescribed above.

The term “LATV” or “live attenuated tetravalent dengue vaccine” or “LATVvaccine” refers to a vaccine comprising an effective amount of a DEN1LAV or LACV, a DEN2 LAV or LACV, a DEN3 LAV or LACV and a DEN4 LAV orLACV. In one embodiment, at least one of the dengue LAVs or LACVscomprises the Δ30 mutation of the TL-2 structure in the 3′ UTR, asdescribed above and in WO 03/092592. In some preferred embodiments, theLATV comprises the following features: (1) rDEN1Δ30, which is a DENV1LAV wherein the DENV1 viral genome comprises a 30 nt deletioncorresponding to the TL2 stem-loop structure in the 3′ UTR; (2)rDEN2/4Δ30, which is a DENV2 LACV comprising the DENV2 prM and E geneson a DENV4 backbone, wherein the DEN4 backbone comprises a 30-ntdeletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (3)rDEN3Δ30/Δ31, which is a DENV3 LAV wherein the DENV3 viral genomecomprises a 30 nt deletion corresponding to the TL2 stem-loop structurein the 3′ UTR and a separate, noncontiguous, upstream 31 nt deletioncorresponding to the TL-3 structure of the 3′ UTR; and (4) rDEN4Δ30,which is a DENV4 LAV wherein the DENV4 viral genome comprises a 30 ntdeletion corresponding to the TL2 stem-loop structure in the 3′ UTR (seeFIG. 1 of WO2016106107).

“Non-replicating vaccine” refers to a dengue virus vaccine for theprevention or treatment of dengue virus infection or the clinicalsymptoms thereof, selected from a recombinant subunit vaccine, aninactivated vaccine, a conjugate vaccine, or a DNA vaccine.

“Inactivated vaccine” refers to a vaccine comprising an effective amountof a killed or inactive whole dengue virus and a pharmaceuticallyacceptable carrier, wherein the virus is inactivated by any means,including with chemicals, heat or radiation. An inactivated vaccine hasa low residual infectivity following inactivation, e.g. <5 plaqueforming units (PFU's)/mL after inactivation. In preferred embodiments,there is very low amount of residual infectivity following inactivation,e.g. ≤4 PFU's/mL, ≤3 PFU's/mL, or ≤2 PFU's/mL, <1 PFU/mL, ≤0.5 PFU/mL,or ≤0.1 PFU/mL. The PFU's of a particular vaccine may be determined, forexample, by using a plaque assay, an immunostaining assay, or othermethod known in the art for detecting viral infectivity.

“Conjugate vaccine” refers to a vaccine comprising a dengue antigencovalently attached to a carrier protein.

A “DNA vaccine” is a vaccine comprising a sequence of nucleotides thatencodes a dengue protein antigen, including dengue proteins, dengueprotein fragments, and dengue fusion proteins, and variants thereof. DNAvaccines comprise a plasmid (e.g. a DNA or viral plasmid) comprising asequence of nucleotides that encode an antigen of interest, operablylinked to a promoter.

“Subunit vaccine” refers to a vaccine that includes one or more dengueantigen components, but not complete dengue viruses, such as dengueimmunogenic epitopes, dengue proteins, dengue antigen fusion proteins,including fusions of different dengue serotype antigens, or dengueprotein fragments. Subunit vaccines, as used herein, can be monovalent(comprise a single dengue antigen) or multivalent (comprise more thanone antigen component). In preferred embodiments, the subunit vaccine istetravalent.

The term “prime-boost” refers to a therapeutic regimen comprising (1)administration to a patient in need thereof a first dengue virus vaccinecomposition, wherein the composition comprises (a) at least one liveattenuated dengue virus (LAV) or live attenuated chimeric flavivirus(LACV), and (b) a pharmaceutically acceptable carrier; (2) waiting for apredetermined amount of time to pass; and (3) administration to thepatient of a second dengue virus vaccine composition or non-replicatingdengue vaccine. The second dengue virus vaccine composition can be thesame or different from the first dengue virus vaccine composition. Inone embodiment, the second dengue virus vaccine is a live attenuateddengue vaccine or a recombinant dengue subunit vaccine. The dengue virusvaccines used in the compositions of the invention are useful forinducing a virus neutralizing antibody response to the homologous dengueviruses in human patients.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Individuals or patients “in needof” treatment include those already with a dengue infection, whether ornot manifesting any clinical symptoms, as well as those at risk of beinginfected with dengue. Treatment of a patient with the dengue vaccinecompositions of the invention includes one or more of the following:inducing/increasing an immune response against dengue in the patient,inducing a virus neutralizing antibody response against one or moredengue viruses, preventing, ameliorating, abrogating, or reducing thelikelihood of the clinical manifestations of dengue in patients who havebeen infected with dengue, preventing or reducing the likelihood ofdeveloping dengue fever, DHF, or DSS and/or other disease orcomplication associated with dengue infection, reducing the severity orduration of the clinical symptoms of dengue infection and/or otherdisease or complication associated with dengue, and preventing orreducing the likelihood of dengue infection.

The term “pharmaceutically effective amount” or “effective amount” meanssufficient vaccine composition is introduced to a patient to produce adesired effect, including, but not limited to: inducing/increasing animmune response against dengue in the patient, inducing/increasing avirus neutralizing antibody response against dengue in a patient,preventing or reducing the likelihood of dengue infection, preventing orreducing the likelihood of dengue recurrent infection, preventing,ameliorating or abrogating the clinical manifestations of dengueinfection in patients who have been infected with dengue, preventingdengue fever, DHF and/or DSS, or reducing the severity or duration ofdisease associated with dengue. One skilled in the art recognizes thatthis level may vary.

The term “immune response” refers to a cell-mediated (T-cell) immuneresponse and/or an antibody (B-cell) response.

The term “patient” refers to a mammal capable of being infected with adengue virus, such as DEN1, DEN2, DEN3, or DEN4, that is to receive thedengue vaccine compositions described herein, including bothimmunocompetent and immunocompromised individuals. In preferredembodiments, the patient is a human. As defined herein, a “patient”includes those already infected with dengue, either through naturalinfection or vaccination or those that may subsequently be exposed.

An “ISCOM-like adjuvant” is an adjuvant comprising an immune stimulatingcomplex (ISCOM), which is comprised of a saponin, cholesterol, and aphospholipid, which together form a characteristic caged-like particle,having a unique spherical, caged-like structure that contributes to itsfunction (for review, see Barr and Mitchell, Immunology and Cell Biology74: 8-25 (1996)). This term includes both ISCOM™ adjuvants, which areproduced with an antigen and comprise antigen within the ISCOM™ particleand ISCOM™ matrix adjuvants, which are hollow ISCOM-type adjuvants thatare produced without antigen. In preferred embodiments of thecompositions and methods provided herein, the ISCOM-type adjuvant is anISCOM™ matrix particle adjuvant, such as ISCOMATRIX™, which ismanufactured without antigen (ISCOM™ and ISCOMATRIX™ are registeredtrademarks of CSL Limited, Parkville, Australia).

The designation “rDEN1Δ30-1545” refers to a recombinant dengue 1 viruswherein the viral genome comprises (1) a 30 nt deletion of the TL2stem-loop structure of the 3′ UTR and (2) a substitution at nucleotideposition 1545 to G, which occurred after adaptation of the virus togrowth in Vero cells.

The designation “rDEN2/4 Δ 30(ME)-1495,7163” refers to a recombinantchimeric dengue 2/4 virus, wherein the viral genome comprises: (1) adengue 4 backbone (C, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5 genes)comprising (i) a 30 nt deletion of the TL2 stem-loop structure of the 3′UTR, and (ii) substitutions at nucleotide position 1495 to U and 7163 toC, which occurred after adaptation of the virus to growth in Vero cells,and (2) dengue 2 prM and E genes.

The designation “rDEN3Δ30/31-7164” refers to a recombinant dengue 3virus wherein the viral genome comprises: (1) a 30 nt deletion of theTL2 stem-loop structure of the 3′ UTR, (2) a separate, 31 nt deletion inthe 3′UTR, upstream of the Δ30 mutation, that deletes the TL-3 structureand (3) a substitution at nucleotide position 7164 to C, which occurredafter adaptation of the virus to growth in Vero cells.

The designation “rDEN4Δ 30-7132,7163,8308” refers to a recombinantdengue 4 virus wherein the viral genome comprises: (1) a 30 nt deletionof the TL2 stem-loop structure of the 3′ UTR and (2) substitutions atnucleotide position 7132 to U, 7163 to C and 8308 to G, which occurredafter adaptation of the virus to growth in Vero cells.

“V180” refers to a tetravalent subunit vaccine comprised of truncatedenvelope glycoproteins (DEN-80E) from each of the 4 dengue virusserotypes (DENV1, DENV2, DENV3, and DENV4), wherein the E proteins eachconstitute approximately 80% of the length of wild type E starting fromamino acid residue 1 at its N-terminus, such that said E protein issecretable into growth medium when expressed recombinantly in a hostcell. See Coller et al. WO 2012/154202.

The following abbreviations are used herein and have the followingmeanings: C is the dengue capsid gene, DEN (alternatively DENV) isdengue virus, DF is dengue fever, DHF is dengue hemorrhagic fever, DSSis dengue shock syndrome, h is hours, GMT is geometric mean titer, IM isintramuscular, IMX is Iscomatrix™, JE is Japanese encephalitis, LAV islive attenuated virus, NS (used in NS1-NS5) is non-structural, nt isnucleotide, PFU is plaque forming units, prM is the dengue preMembranegene, SC is subcutaneous, TBE is tick-borne encephalitis, UTR isuntranslated region, WN (alternatively WNV) is West Nile Virus, YF(alternatively YFV) is yellow fever virus, and wt is wild type.

Live Attenuated Dengue Virus Vaccine

As stated above, the dengue virus vaccine compositions of the inventioncomprise a live attenuated dengue vaccine comprising at least one LAV,selected from the group consisting of dengue virus type 1 (DEN1), denguevirus type 2 (DEN2), dengue virus type 3 (DEN3) and dengue virus type 4(DEN4), or LACV. In one embodiment, the LAV or LACV comprises a viralgenome that comprises a TL-2 Δ30 modification in the 3′UTR, and whereinthe LAV or LACV: induces an immune response against dengue, induces avirus neutralizing antibody response against dengue, protects against orreduces the likelihood of infection or reduces the severity or durationof the clinical manifestations thereof. In embodiments of the invention,the live attenuated dengue vaccine is monovalent, bivalent, trivalent ortetravalent, i.e. induces an immune response against or protects againstone, two, three or four of DEN serotypes 1-4, respectively. In preferredembodiments of the invention, the live attenuated dengue vaccine istetravalent, i.e. induces an immune response against or protects againstDEN serotypes 1-4 and comprises a DEN1, a DEN2, a DEN3 and a DEN4component, wherein each component is either an LAV or an LACV.

In additional embodiments of the invention, the live attenuated denguevaccine is a tetravalent LAV or “LATV” (i.e. comprises live attenuateddengue viruses from DENV 1-4, or live attenuated chimeric flavivirusesfrom DENV 1-4, as defined herein, or a combination thereof, wherein atleast one of the LAVs or LACVs is a Δ30LAV or a Δ30LACV). In additionalembodiments of the invention, the live attenuated dengue vaccine istetravalent and comprises at least one chimeric flavivirus; wherein thechimeric flavivirus comprises a viral genome that contains nucleotidesequences encoding the prM and E proteins of a single dengue virusserotype and nucleotide sequences encoding the capsid and non-structuralproteins of a different flavivirus, wherein the chimeric flavivirus isattenuated. In some embodiments of the invention, the capsid andnonstructural proteins of the chimeric flavivirus is from a differentdengue serotype than the prM and E proteins.

In some embodiments of the invention, each LAV or LACV component of aLATV of the invention comprises a live attenuated virus which isindependently either an attenuated chimeric flavivirus or an attenuateddengue virus comprising the TL-2 Δ30 modification in the 3′UTR of theviral genome. Attenuation of the dengue virus is achieved through theTL-2 Δ30 modification. However, additional attenuating mutations mayalso be included in one or more components of the vaccine, including,but not limited to: mutations at positions 1495, 1545, 7132, 7163, 7164and 8308. Attenuating mutations can be achieved by different techniques,including methods known in the art such as through serial passage ontissue culture or through more defined genetic manipulations. Mutationsuseful for attenuating dengue viruses and chimeric dengue viruses areknown in the art. See, e.g. WO 02/095075, WO 2006/44857, U.S. Pat. Nos.7,189,403, 8,337,860, WO 2003/103571, WO 2000/014245, and WO2008/022196. Known attenuated dengue strains can also be used in thecompositions herein, such as the strains described in WO 06/134433, WO2006/134443, WO 2007/141259, WO 96/40933, WO 2000/057907, WO2000/057908, WO 2000/057909, WO 2000/057910, and WO 2007/015783.

Preferred embodiments of the compositions of the invention comprise atetravalent live attenuated dengue vaccine (LATV). Such tetravalent liveattenuated vaccine can comprise four attenuated dengue viruses (LAVs),three LAVs and one attenuated chimeric flavivirus strain (LACV), twodengue LAVs and two LACVs, one dengue LAV and three LACVs, or fourLACVs.

In preferred embodiments, the LATV comprises the following features: (1)rDEN1Δ30, which is a DENV1 LAV wherein the DENV1 viral genome comprisesa 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′UTR; (2) rDEN2/4Δ30, which is a DENV2 LACV comprising the DENV2 prM andE genes on a DENV4 backbone, wherein the DEN4 backbone comprises a 30-ntdeletion corresponding to the TL2 stem-loop structure in the 3′ UTR; (3)rDEN3 Δ30/Δ31, which is a DENV3 LAV wherein the DENV3 viral genomecomprises a 30 nt deletion corresponding to the TL2 stem-loop structurein the 3′ UTR and a separate, noncontiguous, upstream 31 nt deletioncorresponding to the TL-3 structure of the 3′ UTR; and (4) rDEN4Δ30,which is a DENV4 LAV wherein the DENV4 viral genome comprises a 30 ntdeletion corresponding to the TL2 stem-loop structure in the 3′ UTR.

In embodiments of the invention comprising chimeric flaviviruses, eachchimeric flavivirus comprises a viral genome that comprises nucleotidesequences encoding the prM and E proteins of a single dengue virusserotype and nucleotide sequences that encode the capsid andnon-structural proteins (i.e. “the backbone”) of a different flavivirus,wherein each of the chimeric flaviviruses are attenuated. Methods forconstruction of a recombinant live attenuated flavivirus strain maycomprise the use of a known attenuated strain as a base, wherein themethod comprises substituting the appropriate genes (prM and E) from arelated virus of interest for the equivalent genes of the base virus.For example, this approach has been used for WNV wherein the chimericvirus is an intertypic chimeric based on an attenuated DEN-4 straincomprising prM and E genes of WNV (Bray, M. et al., J. Virol. (1996)70:4162-4166; Chen, W., et al., J Virol. (1995) 69:5186-5190; Bray, M.and Lai, C.-J., Proc. Natl. Acad. Sci. USA (1991) 88:10342-10346; Lai,C. J. et al., Clin. Diagn. Virol. (1998) 10:173-179).

Another approach has been the use of the YF 17D attenuated yellow feverstrain as a base to develop recombinant chimeric vaccines, which waspreviously used for JE virus, DEN viruses, and WN virus. A chimericyellow fever vaccine can be constructed comprising a yellow feverbackbone by replacing the genes coding for prM and E proteins from anyyellow fever strain, for example, YFV 17D, with those of a Dengueserotype. After DNA cloning, RNA is transcribed and transfected intoVero cells to obtain chimeric viruses possessing the YFV 17D replicationmachinery and the external coat of the relevant Dengue virus. SeeGuirakhoo et al., Journal of Virology, 74(12): 5477-5485 (2000); Guy etal., Vaccine 28: 632-649 (2010); Monath T. P. Adv Virus Res (2003)61:469-509; Monath et al. Proc. Natl. Acad. Sci. USA (2006) 103:6694;and WO 98/37911. Thus, in some embodiments of the invention, the liveattenuated dengue vaccine comprises (1) at least one chimeric flaviviruscomprising the prM and E proteins of a single dengue serotype and ayellow fever backbone and (2) at least one LAV or LACV which comprises aviral genome comprising a 30-nucleotide deletion of the TL-2 stem-loopstructure of the 3′UTR.

Chimeric live attenuated flaviviruses useful in the compositions of theinvention may also comprise a dengue chimeric virus, wherein the viralgenome comprises prM and E genes of a single dengue virus serotype andthe capsid and nonstructural genes of a different dengue virus serotype.In embodiments wherein the chimeric virus comprises a backbone from asecond dengue serotype, the dengue backbone comprises a deletion ofabout 30-nucleotides of the 3′UTR that corresponds to the TL-2 stem-loopstructure and may optionally comprise additional attenuating mutations.Any attenuated dengue virus or wild-type dengue virus can be used as thebackbone of the chimeric virus, by introduction of a 30-nucleotidedeletion of the TL-2 stem-loop structure to an attenuated denguebackbone or wild-type dengue viral backbone. Attenuation of a denguevirus backbone can be achieved through serial passage, through theintroduction of defined genetic mutations, or through the use of knownattenuated dengue strains. Dengue chimeric vaccines are described, forexample, in Whitehead et al. WO 03/092592. In some embodiments of theinvention, the live attenuated vaccine comprises a chimeric flaviviruswherein the capsid and nonstructural proteins are from a differentdengue serotype than the prM and E proteins.

The dengue virus vaccine compositions of the invention comprise aneffective amount of live attenuated virus vaccine. In some embodimentsof the invention, the potency of the live attenuated dengue vaccine isfrom 10 to about 1×10⁷ plaque forming units (PFU's). In alternativeembodiments, the potency of the live attenuated dengue vaccine is fromabout 1×10² to about 1×10⁶ PFU's. In other embodiments, the potency ofthe live attenuated dengue vaccine is from about 1×10³ to about 1×10⁵PFU's.

Viral plaque assays determine the number of plaque forming units (pfu)in a virus sample. Briefly, in a dengue immunoplaque assay, a confluentmonolayer of host cells (e.g., Vero cells) is infected with dengue virusat varying dilutions and covered with a semi-solid overlay medium,containing methylcellulose, to prevent the virus infection fromspreading indiscriminately. The virus infected cell(s) will lyse andspread the infection to adjacent cells where the infection-to-lysiscycle is repeated. The infected cells will form a plaque (a group ofinfected Vero cells surrounded by uninfected cells) which can be seenvisually after fixing and immune-staining using anti dengue serotypespecific monoclonal antibodies (mAb). Plaques are counted and theresults, in combination with the dilution factors, are used to calculatethe number of plaque forming units per mL (pfu/mL) in the samples. Thedengue potency result in pfu/mL represents the number of infectiousparticles within the sample and is based on the assumption that eachplaque formed is representative of one infectious virus particle.

Dengue Subunit Vaccine

In some embodiments of the invention, the formulations further comprisea recombinant dengue subunit vaccine which comprises one or more dengueantigen proteins. In preferred embodiments of this aspect of theinvention, the recombinant dengue subunit vaccine comprises one or moredengue proteins, fusion proteins, or a fragment or fragments thereof. Infurther preferred embodiments, the recombinant dengue subunit vaccinecomprises dengue envelope or E protein, or a fragment thereof.

In further preferred embodiments, the recombinant dengue subunit vaccineis tetravalent, i.e. targets an immune response against all four dengueserotypes. A recombinant dengue subunit vaccine can comprise fourrecombinant dengue proteins or less than four, e.g. a recombinant DEN1protein, a recombinant DEN2 protein, and a recombinant DEN3/4 fusionprotein. In some embodiments, the recombinant dengue subunit vaccinecomprises dengue virus envelope glycoprotein, or fragments thereof, ofDEN1-4 (e.g. DEN1-80E, DEN2-80E, DEN3-80E, DEN4-80E, or DEN4-80EZip)that is produced and secreted using a recombinant expression system.Said subunit vaccine may optionally comprise an adjuvant, as describedmore fully below.

In some embodiments of this aspect of the invention, the recombinantdengue subunit vaccine comprises one or more purified dengue virusenvelope (“E”) proteins, a pharmaceutically acceptable excipient,wherein the E proteins each constitute approximately 80% of the lengthof wild type E starting from amino acid residue 1 at its N-terminus,such that said E protein is secretable into growth medium when expressedrecombinantly in a host cell and wherein the composition induces theproduction of neutralizing antibodies in human subjects. In someembodiments of the invention, the recombinant dengue subunit vaccinefurther comprises an effective amount of an adjuvant. In someembodiments of the invention, the DEN-4 E protein is dimeric(“DEN4-80EZip”), as described in U.S. Pat. No. 6,749,857 and WO2012/154202.

In some embodiments of this aspect of the invention, the E proteins inthe composition described above are recombinantly produced and expressedin insect host cells. In further preferred embodiments, the E protein isrecombinantly produced and expressed in Drosophila melanogasterSchneider 2 (S2) host cells.

The recombinant dengue virus E proteins of can be produced by means of acell culture expression system that uses Drosophila Schneider 2 (S2)cells. This system has been demonstrated to produce recombinant dengueenvelope proteins that maintain native-like structure (Cuzzubbo et al.,Clin. Diagn. Lab. Immunol. (2001) 8:1150-55; Modis et al., Proc. Natl.Acad. Sci. (2003) 100:6986-91; Modis et al., Nature (2004) 427:313-9;Zhang et al., Structure (2004)₁₂(9):1607-18). This expression system hasalso been shown to express other recombinant envelope proteins fromother flaviviruses such as West Nile, Japanese Encephalitis, hepatitisC, and Tick Borne Encephalitis viruses. The recombinant dengue envelopeproteins may be truncated at the C-terminus, leaving 80% of the nativeenvelope protein (“80E”). Thus 80E is defined as approximately the first80% of consecutive amino acids of E protein starting at amino acid 1 ofits N-terminus.

As stated above, some embodiments of this aspect of the inventioncomprise truncated 80E proteins which consist of approximately 80% ofthe length of wild type E starting from amino acid residue 1 at itsN-terminus. The E proteins used in some embodiments of the inventiondelete the membrane anchor portion (approximately the last 10% of E atthe carboxy end) of the protein. In other words, truncated 80E proteinsof use in specific embodiments of the invention consist of up to thefirst 90% of consecutive amino acids of E starting at amino acid 1 ofits N-terminus, thus allowing it to be secreted into the extracellularmedium, facilitating recovery. The truncation may further delete the“stem” portion of the E protein that links the 80E portion with themembrane anchor portion; the stem portion does not contain notableantigenic epitopes and therefore is not included in the preferredantigens, DEN1-80E, DEN2-80E, DEN3-80E, DEN4-80E, or DEN4-80EZip. Morethan 90%, but less than 100%, of the E protein can be cloned andsecreted, i.e., the protein can be 90%+ in length, carboxy truncated,and can include a portion of the membrane spanning domain so long as thetruncated E protein is secretable. “Secretable” means able to besecreted, and typically secreted, from the transformed cells in theexpression system. Thus, one of skill in the art will realize thatdengue E proteins that are useful in the compositions and methods of thepresent invention may vary from the 80% exemplified herein, as long asthe protein is secretable. In preferred embodiments of each aspect ofthe present invention, the DEN E proteins are about 80% in lengthstarting from the N-terminal amino acid of the envelope protein andending at an amino acid in the range of the 393^(rd) to 401^(st) aminoacid, for example, from amino acid 1 to amino acid 395 of dengue virustype 2. In alternative embodiments of each aspect of the invention, thedengue E protein may be about 75%, about 85%, about 90%, about 95%, orabout 98% of the consecutive amino acids of E starting at amino acid 1of its N-terminus. In exemplary embodiments of aspects of the inventionherein, the DEN E protein is approximately 80% of consecutive aminoacids of E protein starting at amino acid 1 of its N-terminus; such asDEN1-80E, as set forth in SEQ ID NO:1, DEN2-80E, as set forth in SEQ IDNO:2, DEN3-80E, as set forth in SEQ ID NO:3 and DEN4-80E, as set forthin SEQ ID NO:4.

The secreted E protein may further contain domains which facilitatedimerization, such as in the DEN4-80EZip protein, such that theimmunogenicity of the recombinant protein is further enhanced. Anexemplary DEN4-80EZip protein comprises an amino acid sequence as setforth in SEQ ID NO:5. In some embodiments of this aspect of theinvention, the DEN1, DEN2, and DEN3 80E antigens included in thecomposition are monomeric and the DEN4 80E antigen is dimeric.

In alternative embodiments of this aspect of the invention, theDEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E proteins in the compositionare monomeric. In such embodiments, the DEN4 component is present in anamount that is about 1.5 to about 3 times the individual amounts ofDEN1, DEN2, and DEN3 proteins, preferably about 2 times the amount ofthe DEN1, DEN2, and DEN3 components (proteins). In preferred embodimentsof this aspect of the invention, the ratio of DEN1:DEN2:DEN3:DEN4antigens in the compositions is approximately 1:1:1:2.

In embodiments of the invention comprising dengue E proteins, the amountof each E protein in the composition is from about 0.5 μg to about 500μg. In alternative embodiments, the amount of each E protein is fromabout 0.5 μg to about 450 μg, 0.5 μg to about 400 μg, 0.5 μg to about350 μg, 0.5 μg to about 300 μg, 0.5 μg to about 250 μg, 0.5 μg to about200 μg, 0.5 μg to about 150 μg, 0.5 μg to about 100 μg, 0.5 μg to about50 μg, 5.0 μg to about 500 μg, 5.0 μg to about 450 μg, 5.0 μg to about400 μg, 5.0 μg to about 350 μg, 5.0 μg to about 300 μg, 5.0 μg to about250 μg, 5.0 μg to about 200 μg, 5.0 μg to about 150 μg, 5.0 μg to about100 μg, 5.0 μg to about 50 μg, 10 μg to about 500 μg, 10 μg to about 450μg, 10 μg to about 400 μg, 10 μg to about 350 μg, 10 μg to about 300 μg,10 μg to about 250 μg, 10 μg to about 200 μg, 10 μg to about 150 μg, 10μg to about 100 μg, 10 μg to about 50 μg, 25 μg to about 500 μg, 25 μgto about 450 μg, 25 μg to about 400 μg, 25 μg to about 350 μg, 25 μg toabout 300 μg, 25 μg to about 250 μg, 25 μg to about 200 μg, 25 μg toabout 150 μg, 25 μg to about 100 μg, or 25 μg to about 50 μg. In furtherpreferred embodiments, the amount of each E protein in the compositionis from about 1.0 μg to about 100 μg. In still further embodiments, theamount of each E protein in the composition is selected fromapproximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, or 500 μg.

Inactivated Dengue Vaccine

Inactivated dengue vaccines herein comprise one or more wholeinactivated dengue viruses and/or one or more inactivated denguechimeric viruses. In some embodiments of this aspect of the invention,the inactivated dengue vaccine is tetravalent and comprises wholeinactivated DEN1, DEN2, DEN3 and DEN4. In alternative embodiments, theinactivated vaccine comprises four inactivated chimeric dengue viruses.In still other embodiments, the inactivated vaccine is tetravalent andcomprises one or more whole inactivated dengue viruses and one or moreinactivated dengue chimeric viruses, e.g. an inactivated whole DEN1virus, an inactivated whole DEN2 virus, an inactivated DEN3 chimericvirus and an inactivated DEN4 chimeric virus. One of skill in the artrealizes that any combination of inactivated whole or chimeric DENviruses may be used in the tetravalent compositions and methods of theinvention, as long as the vaccine composition targets all four dengueserotypes.

Inactivated dengue vaccines useful in the compositions and methods ofthe invention are described in Putnak et al. Vaccine 23: 4442-4452(2005), U.S. Pat. Nos. 6,190,859, 6,254,873 and Sterner et al. WO2007/002470. Alternatively, dengue virus strains and chimeric denguestrains/chimeric flavivirus strains can be inactivated for use in thecompositions through methods known in the art, e.g., with chemicals,heat or radiation.

Accordingly, the present invention also relates to the aboveformulations comprising effective amounts of a live attenuated denguevaccine and a non-replicating dengue vaccine, wherein the live,attenuated dengue vaccine comprises at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus (LACV),wherein the LAV or LACV comprise a viral genome that comprises a30-nucleotide deletion of the TL-2 stem-loop structure in the 3′UTR. Insome embodiments of the invention, the non-replicating dengue vaccine ofthe dengue virus vaccine compositions of the invention are selected froma recombinant dengue subunit vaccine or an inactivated dengue vaccine.In one embodiment, the formulation is lyophilized, frozen, microwavedried or has lyospheres with effective amounts of a live attenuateddengue vaccine and a non-replicating dengue vaccine. In anotherembodiment, the formulation of live attenuated dengue vaccine isreconstituted with a liquid solution comprising the non-replicatingdengue vaccine, for example V180.

In preferred embodiments of the invention, the live attenuated and thenon-replicating dengue vaccines are tetravalent (i.e. comprise DEN1,DEN2, DEN3, and DEN4 components or induce an immune response againstDEN1, DEN2, DEN3, and DEN4).

Adjuvants

Co-administration of vaccines with compounds that can enhance the immuneresponse against the antigen of interest, known as adjuvants, has beenextensively studied. In addition to increasing the immune responseagainst the antigen of interest, some adjuvants may be used to decreasethe amount of antigen necessary to provoke the desired immune responseor decrease the number of injections needed in a clinical regimen toinduce a durable immune response and provide protection from disease.

To that end, the dengue virus vaccine formulations of the invention mayemploy an adjuvant. The adjuvant of the formulations described hereincan be any adjuvant that performs the desired function, as describedabove, and does not inactivate or significantly impact the titer of theLAV or LACV of the composition.

Aluminum-based compounds were determined to possess adjuvant activityover 60 years ago (for review, see Lindblad, E. B. Immunol. and CellBiol. 82: 497-505 (2004); Baylor et al. Vaccine 20: S18-S23 (2002)).Aluminum adjuvants are generally regarded as safe when used atappropriate dosages. Many have been approved for administration intohumans by regulatory agencies worldwide.

Accordingly, aluminum-based compounds, such as aluminum hydroxide(Al(OH)₃), aluminum hydroxyphosphate (AlPO₄), amorphous aluminumhydroxyphosphate sulfate (AAHS), or so-called “alum” (KAl(SO₄).12H₂O)(see Klein et al., Analysis of aluminum hydroxyphosphate vaccineadjuvants by Al MAS NMR., J. Pharm. Sci. 89(3): 311-21 (2000)), may becombined with the compositions provided herein. In exemplary embodimentsof the invention provided herein, the aluminum adjuvant is aluminumhydroxyphosphate or AAHS. In alternative embodiments, the aluminumadjuvant is an aluminum phosphate adjuvant, referred to herein as “APA”.In other embodiments, the adjuvant is aluminum hydroxide.

One of skill in the art will be able to determine an optimal dosage ofaluminum adjuvant that is both safe and effective at increasing theimmune response to the targeted dengue viruses. For a discussion of thesafety profile of aluminum, as well as amounts of aluminum included inFDA-licensed vaccines, see Baylor et al., Vaccine 20: S18-S23 (2002).Generally, an effective and safe dose of aluminum adjuvant varies from50 μg to 1.25 mg elemental aluminum per dose (100 μg/mL to 2.5 mg/mLconcentration).

Thus, specific embodiments of the present invention include compositionscomprising a live attenuated dengue virus vaccine and further comprisingan aluminum adjuvant. In embodiments of the invention, the denguecompositions comprise an adjuvant which comprises from about 50 μg toabout 1.25 mg of elemental aluminum per dose of vaccine. In otherembodiments, the aluminum adjuvant per dose of vaccine compositioncomprises an amount of elemental aluminum ranging from about 100 μg toabout 1.0 mg, from about 100 μg to about 900 μg, from about 100 μg toabout 850 μg, from about 100 μg to about 800 μg, from about 100 μg toabout 700 μg, from about 100 μg to about 600 μg, from about 100 μg toabout 500 μg, from about 100 μg to about 400 μg, from about 100 μg toabout 300 μg, from about 100 to about 250 μg, from about 200 μg to about1.25 mg, from about 200 μg to about 1.0 mg, from about 200 μg to about900 μg, from about 200 μg to about 850 μg, from about 200 μg to about800 μg, from about 200 μg to about 700 μg, from about 200 μg to about600 μg, from about 200 μg to about 500 μg, from about 200 μg to about400 μg, from about 200 μg to about 300 μg, from about 300 μg to about1.25 mg, from about 300 μg to about 1.0 mg, from about 300 μg to about900 μg, from about 300 μg to about 850 μg, from about 300 μg to about800 μg, from about 300 μg to about 700 μg, from about 300 μg to about600 μg, from about 300 μg to about 500 μg, from about 300 μg to about400 μg, from about 400 μg to about 1.25 mg, from about 400 μg to about1.0 mg, from about 400 μg to about 900 μg, from about 400 μg to about850 μg, from about 400 μg to about 800 μg, from about 400 μg to about700 μg, from about 400 μg to about 600 μg, from about 400 μg to about500 μg, from about 500 μg to about 1.25 mg, from about 500 μg to about1.0 mg, from about 500 μg to about 900 μg, from about 500 μg to about850 μg, from about 500 μg to about 800 μg, from about 500 μg to about700 μg, from about 500 μg to about 600 μg, from about 600 μg to about1.25 mg, from about 600 μg to about 1.0 mg, from about 600 μg to about900 μg, from about 600 μg to about 850 μg, from about 600 μg to about800 μg, or from about 600 μg to about 700 μg.

Other adjuvants that may be used in conjunction with the dengue virusvaccine compositions of the invention, include, but are not limited to,adjuvants containing CpG oligonucleotides, or other molecules acting ontoll-like receptors such as TLR4 and TLR9 (for reviews, see,Daubenberger, C. A., Curr. Opin. Mol. Ther. 9(1):45-52 (2007); Duthie etal., Immunological Reviews 239(1): 178-196 (2011); Hedayat et al.,Medicinal Research Reviews 32(2): 294-325 (2012)), includinglipopolysaccharide, monophosphoryl lipid A, and aminoalkyl glucosaminide4-phosphates. Additional adjuvants useful in the compositions of theinvention include immunostimulatory oligonucleotides (IMO's; see, e.g.U.S. Pat. Nos. 7,713,535 and 7,470,674); T-helper epitopes, lipid-A andderivatives or variants thereof, liposomes, calcium phosphate,cytokines, (e.g. granulocyte macrophage-colony stimulating factor(GM-CSF) IL-2, IFN-α, Flt-3L), CD40, CD28, CD70, IL-12, heat-shockprotein (HSP) 90, CD134 (OX40), CD137, CoVaccine HT, non-ionic blockcopolymers, incomplete Freund's adjuvant, chemokines, cholera toxin; E.coli heat-labile enterotoxin; pertussis toxin; muramyl dipeptide,muramyl peptide analogues, MF59, SAF, immunostimulatory complexes,biodegradable microspheres, polyphosphazene; synthetic polynucleotides.

Additional adjuvants for use with the compositions described herein areadjuvants containing saponins (e.g. QS21), either alone or combined withcholesterol and phospholipid in the characteristic form of an ISCOM(“immune stimulating complex,” for review, see Barr and Mitchell,Immunology and Cell Biology 74: 8-25 (1996); and Skene and Sutton,Methods 40: 53-59 (2006)). Such adjuvants are referred to herein as“saponin-based adjuvants”. In specific embodiments of the compositionsherein, the mutant toxins and/or toxin proteins are combined with anISCOM-type adjuvant or “ISCOM”, which is an ISCOM matrix particleadjuvant, such as ISCOMATRIX™, which is manufactured without antigen(ISCOM™ and ISCOMATRIX™ are the registered trademarks of CSL Limited,Parkville, Australia).

Formulations

The formulations or compositions of the invention comprise a liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus (LACV),a buffer at pH about 6.5 to 8.5, a sugar, a glycol or sugar alcohol, anda cellulose derivative selected from the group consisting ofcarboxymethyl cellulose, hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), 2-hydroxyethyl cellulose (2-HEC),crosscarmellose, and methyl cellulose, or a pharmaceutically acceptablesalt thereof optionally an alkali or alkaline salt, and optionally anamino acid selected from the group consisting of Ala, Asp, His, Leu,Lys, Gln, Pro or Glu, or a combination thereof.

In another aspect of the invention, the formulation comprises liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus at about20-200,000,00 pfu/ml, a buffer at pH about 6.5 to 8.5, a sugar at about150-300 mg/ml, a carrier selected from the group consisting ofpolyvinylpyrrolidone (PVP), carboxymethyl cellulose, hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethylcellulose (2-HEC), crosscarmellose, methyl cellulose or apharmaceutically acceptable salt thereof, Human Serum Albumin (HSA) andgelatin; optionally an alkali salt or alkaline salt at about 5-100 mM;and optionally an amino acid Gln, Pro or Glu, or a combination thereof.

In one embodiment, the live attenuated dengue vaccine is at aconcentration of 100-10,000,000 pfu/ml, 100-100,000 pfu/ml, or600-20,000 pfu/ml in the formulation. In another embodiment, the liveattenuated dengue vaccine is at a concentration of 200-200,000 pfu/ml,600-200,000 pfu/ml, or 600-100,000 pfu/ml in the formulation.

In preferred embodiments, the cellulose derivative is anionic and formsa salt, for example carboxymethyl cellulose sodium or potassium at about0.3-10 mg/ml, 1-10 mg/ml, 3-7 mg/ml or 5 mg/ml in the live attenuateddengue vaccine formulation. Carboxymethyl cellulose salt is available inhigh viscosity type with average molecular weight of about 700,000;medium viscosity type with average molecular weight of about 250,000;and low viscosity type with average molecular weight of about 90,000. Inone embodiment, the cellulose derivative is carboxymethyl cellulose saltwith average molecular weight of about 700,000 at about 0.3-1.5 mg/ml inthe live attenuated dengue vaccine formulation. In another embodiment,the cellulose derivative is carboxymethyl cellulose salt with averagemolecular weight of about 250,000 at about 1-4 mg/ml. In a furtherembodiment, the cellulose derivative is carboxymethyl cellulose saltwith average molecular weight of about 90,000 at about 3-7 or 3-10mg/ml. In yet a further embodiment, the cellulose derivative iscarboxymethyl cellulose salt with average molecular weight of about50,000 to 1000,000 at about 0.3-10 mg/ml.

In one embodiment, the buffer is selected from the group consisting ofphosphate, succinate, histidine, TRIS, MES, MOPS, HEPES, acetate andcitrate at about 5-300 mM, 5-20 mM, 10-12 mM or 11 mM.

The alkali or alkaline salt can provide a stabilizing effect and can beselected

from

the group consisting of magnesium chloride, calcium chloride, potassiumchloride, and sodium chloride or a combination thereof at about 10-150mM, 10-100 mM, 15-75 mM, 30-90 mM, 75 mM, 50 mM or 30 mM.

The amino acid can be selected from the group consisting of Val, Ile,Ala, Asp, His, Leu, Lys, Gln, Pro and Glu, or a combination thereof at10-100, 10-75, 10-50, 20-30, or 25 mM. In another embodiment, the aminoacid can be selected from the group consisting of Ala, Asp, His, Leu,Lys, Gln, Pro and Glu, or a combination thereof at 10-100, 10-75, 10-50,20-30, or 25 mM. In one embodiment, the amino acid is Lys, Leu or Glu.In another embodiment, the amino acids are Leu and Glu. In anotherembodiment, the amino acid is Leu, Lys, Glu, or Ala. In anotherembodiment, the amino acid is Leu.

The sugar and glycol or sugar alcohol can act as a cryoprotectant orstabilizing excipient. In one embodiment, the sugar is at aconcentration of 50-300 mg/ml. In another embodiment, the sugar istrehalose or sucrose or a combination thereof at about 60-120 mg/ml,90-110 mg/ml, or 80-100 mg/ml. In one embodiment, the sucrose totrehalose ratio is between 1:1 to 1:4. In another embodiment, thesucrose is 90 mg/ml and the trehalose is 90-200 mg/ml, and preferably110 mg/ml. In another embodiment, the glycol is propylene glycol, andthe sugar alcohol is glycerol or sorbitol at about 2.5-7.5 mg/ml, 3-7mg/ml or 5 mg/ml.

The compositions of the invention can be administered to a subject byone or more methods known to a person skilled in the art, such asparenterally, transmucosally, transdermally, intramuscularly,intravenously, intra-dermally, intra-nasally, subcutaneously,intra-peritonealy, and formulated accordingly.

In one embodiment, compositions of the present invention areadministered via epidermal injection, intramuscular injection,intravenous, intra-arterial, subcutaneous injection, orintra-respiratory mucosal injection of a liquid preparation. Liquidformulations for injection include solutions and the like. Thecomposition of the invention can be formulated as single dose vials,multi-dose vials or as pre-filled syringes.

In another embodiment, compositions of the present invention areadministered orally, and are thus formulated in a form suitable for oraladministration, i.e., as a solid or a liquid preparation. Solid oralformulations include tablets, capsules, pills, granules, pellets and thelike. Liquid oral formulations include solutions, suspensions,dispersions, emulsions, oils and the like.

In one aspect of the invention, the formulation is a solid driedformulation prepared from lyophilization, freezing, microwave drying orthrough the generation of lyospheres. In one embodiment, the solid driedformulation is obtainable by or produced from the microwave dryingprocess described in example 7. The formulations can be stored at −70°C., −20° C., 2-8° C. or at room temperature (25 or 37° C.). The driedformulations can be expressed in terms of the weight of the componentsin a unit dose vial, but this varies for different doses or vial sizes.Alternatively, the dried formulations of the present invention can beexpressed in the amount of a component as the ratio of the weight of thecomponent compared to the weight of the drug substance (DS) in the samesample (e.g. a vial). This ratio may be expressed as a percentage. Suchratios reflect an intrinsic property of the dried formulations of thepresent invention, independent of vial size, dosing, and reconstitutionprotocol. In other embodiments, the formulation is in lyospheres.

In another aspect of the invention, the formulation is a reconstitutedsolution. A dried solid formulation can be reconstituted at differentconcentrations depending on clinical factors, such as route ofadministration or dosing. For example, a dried formulation may bereconstituted at a high concentration (i.e. in a small volume) ifnecessary for subcutaneous administration. High concentrations may alsobe necessary if high dosing is required for a particular subject,particularly if administered subcutaneously where injection volume mustbe minimized. Subsequent dilution with water or isotonic buffer can thenreadily be used to dilute the drug product to a lower concentration. Ifisotonicity is desired at lower drug product concentration, the driedpowder may be reconstituted in the standard low volume of water and thenfurther diluted with isotonic diluent, such as 0.9% sodium chloride.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, e.g., onthe type of diluent, amount of excipient(s) and virus or protein.Exemplary diluents include sterile water, bacteriostatic water forinjection (BWFI), a pH buffered solution (e.g. phosphate-bufferedsaline), sterile saline solution, Ringer's solution or dextrosesolution. The reconstitution volume can be about 0.5-1.0 ml, preferably0.5 ml or 0.7 ml. In one embodiment, a single dose has a volume of 0.5ml. In another aspect, the invention provides a method of preparing aliquid formulation comprising the steps of reconstituting theformulations of the invention with a diluent as described above.

In another embodiment of the invention, the formulation is the aqueoussolution prepared before lyophilization, freezing, microwave drying orgeneration of lyospheres.

Processes for Preparing the Lyospheres

Processes for preparing lyospheres are disclosed in US patentpublication US20140294872, the disclosure of which is hereinincorporated by reference in its entirety. The method comprisesdispensing at least one liquid droplet having a substantially sphericalshape onto a solid and flat surface (i.e., lacking any sample wells orcavity), freezing the droplet on the surface without contacting thedroplet with a cryogenic substance and lyophilizing the frozen dropletto produce a dried pellet that is substantially spherical in shape. U.S.Pat. No. 9,119,794, the disclosure of which is herein incorporated byreference in its entirety, also discloses processes for forminglyospheres. The unitary volumes containing the aqueous medium mixtureare formed on a solid element containing cavities. The solid element iscooled below the freezing temperature of the mixture, the cavities arefilled with the mixture, and the mixture is solidified while present inthe cavity to form the unitary forms. The unitary forms are dried in avacuum to provide the lyospheres.

In other embodiments, the lyospheres are formed in a substantiallyspherical shape and are prepared by freezing droplets of a liquidcomposition of a desired biological material on a flat, solid surface,in particular, a surface that does not have any cavities, followed bylyophilizing the unitary forms. U.S. Patent Application Publication No.US2014/0294872, the disclosure of which is herein incorporated byreference, discloses similar processes for forming lyospheres.

Briefly, in some embodiments the process comprises dispensing at leastone liquid droplet having a substantially spherical shape onto a solidand flat surface (i.e., lacking any sample wells or cavity), freezingthe droplet on the surface without contacting the droplet with acryogenic substance and lyophilizing the frozen droplet to produce adried pellet that is substantially spherical in shape. The process maybe used in a high throughput mode to prepare multiple dried pellets bysimultaneously dispensing the desired number of droplets onto the solid,flat surface, freezing the droplets and lyophilizing the frozendroplets. Pellets prepared by this process from a liquid formulation mayhave a high concentration of a biological material (such as a proteintherapeutic) and may be combined into a set of dried pellets.

In some embodiments, the solid, flat surface is the top surface of ametal plate which comprises a bottom surface that is in physical contactwith a heat sink adapted to maintain the top surface of the metal plateat a temperature of −90° C. or below. Since the top surface of the metalplate is well below the freezing point of the liquid formulation, thedroplet freezes essentially instantaneously with the bottom surface ofthe droplet touching the top surface of the metal plate.

In other embodiments, the solid, flat surface is hydrophobic andcomprises the top surface of a thin film that is maintained above 0° C.during the dispensing step. The dispensed droplet is frozen by coolingthe thin film to a temperature below the freezing temperature of theformulation.

Lyophilization Process

The lyophilized formulations of the present invention are formed bylyophilization (freeze-drying) of a pre-lyophilization solution.Freeze-drying is accomplished by freezing the formulation andsubsequently subliming water at a temperature suitable for primarydrying. Under this condition, the product temperature is below theeutectic point or the collapse temperature of the formulation.Typically, the shelf temperature for the primary drying will range fromabout −50 to 25° C. (provided the product remains frozen during primarydrying) at a suitable pressure, ranging typically from about 30 to 250mTorr. The formulation, size and type of the container holding thesample (e.g., glass vial) and the volume of liquid will dictate the timerequired for drying, which can range from a few hours to several days(e.g. 40-60 hrs). A secondary drying stage may be carried out at about0-40° C., depending primarily on the type and size of container and thetype of protein employed. The secondary drying time is dictated by thedesired residual moisture level in the product and typically takes atleast about 5 hours. Typically, the moisture content of a lyophilizedformulation is less than about 5%, and preferably less than about 3%.The pressure may be the same as that employed during the primary dryingstep. Freeze-drying conditions can be varied depending on theformulation, vial size and lyophilization trays.

In some instances, it may be desirable to lyophilize or microwave drythe formulation in the container in which reconstitution is to becarried out in order to avoid a transfer step. The container in thisinstance may, for example, be a 2, 3, 5, 10 or 20 ml vial.

Methods of Use

Embodiments of the invention also include one or more of the denguevaccine compositions or formulations described herein (i) for use in,(ii) for use as a medicament or composition for, or (iii) for use in thepreparation of a medicament for: (a) therapy (e.g., of the human body);(b) medicine; (c) inhibition of dengue virus replication, includingDEN1, DEN2, DEN3 and/or DEN4; (d) induction of an immune response or aprotective immune response against one or more of DEN1, DEN2, DEN3and/or DEN4; (e) induction of a virus neutralizing antibody responseagainst one or more types of dengue; (f) treatment or prophylaxis ofinfection by dengue virus; (g) prevention of recurrence of dengue virusinfection; (h) reduction of the progression, onset or severity ofpathological symptoms associated with dengue virus infection and/orreduction of the likelihood of a dengue virus infection or, (i)treatment, prophylaxis of, or delay in the onset, severity, orprogression of dengue-associated disease(s), including, but not limitedto: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome.In these uses, the dengue vaccine compositions can optionally beemployed in combination with one or more adjuvants (e.g., AAHS, aluminumphosphate, aluminum hydroxide such as Alhydrogel®, or other aluminumsalt adjuvant, a saponin-based adjuvant such as ISCOMATRIX™ (CSL, Ltd.),a TLR-agonist, or lipid nanoparticles, described herein).

Prophylactic treatment can be performed using a dengue virus vaccinecomposition of the invention, as described herein. The composition ofthe invention can be administered to the general population or to thosepersons at an increased risk of dengue infection, e.g. those persons wholive in or will be travelling to areas of the world in which mosquitoesof the genus Aedes are prevalent.

Those “in need of treatment” include those already with a dengueinfection (e.g. infected with one or more of DEN1, DEN2, DEN3, or DEN4),as well as those prone to have an infection or any person in which areduction in the likelihood of infection is desired.

Dengue virus vaccine compositions of the invention can be formulated andadministered to a patient using techniques well known in the art.Guidelines for pharmaceutical administration in general are provided in,for example, Vaccines Eds. Plotkin and Orenstein, W.B. Sanders Company,1999; Remington's Pharmaceutical Sciences 20^(th) Edition, Ed. Gennaro,Mack Publishing, 2000; and Modern Pharmaceutics 2^(nd) Edition, Eds.Banker and Rhodes, Marcel Dekker, Inc., 1990.

Accordingly, the invention provides a method for inducing a protectiveimmune response in a patient against a dengue infection comprising thestep of administering to the patient an immunologically effective amountof any of the dengue virus vaccine compositions described herein. In oneembodiment, the dengue virus vaccine composition is co-administered incombination with other vaccines for treating or preventing diseases fromZika, Measles Mumps and Rubella, or Varicella etc.

Also provided by the invention is a method for treating dengueinfection, or for treating any pathological condition associated withdengue infection, such treatment including prophylaxis of infection, andreduction in the severity of clinical symptoms, delay or prevention ofthe progression of disease, and/or reduction in the likelihood ofinfection or the clinical symptoms thereof; the method comprising thestep of administering to the patient an immunologically effective amountof any of the vaccine compositions as described herein.

Additional embodiments of the invention comprise the administration oftwo or more compositions of the invention to a patient in a prime/boostregime. Accordingly, the invention relates to a method of preventing orreducing the likelihood of dengue infection in a patient in needthereof, comprising the steps of:

(a) administering a first dengue virus vaccine composition of theinvention to the patient;

(b) waiting for a predetermined amount of time to pass after step (a);

(c) administering to the patient a second dengue virus vaccinecomposition of the invention; and,

(d) optionally repeating steps (b) and (c);

whereby the dengue infection is prevented or the likelihood of beinginfected with dengue is reduced in the patient.

In embodiments of the method above, the dengue virus vaccinecompositions of the invention are in the form of a frozen liquid. Inalternative embodiments, the dengue virus vaccine compositions arelyophilized, or microwaved dried and reconstituted with a sterilediluent prior to administration to the patient.

The amount of time between the first dose of a dengue virus vaccinecomposition of the invention and the second dose of a dengue virusvaccine composition of the invention, or any dose thereafter, is fromabout 2 weeks to about 2 years. In preferred embodiments of theinvention, a time of 2 months to 12 months is allowed to pass betweenmultiple administrations. In alternative embodiments of this aspect ofthe invention, the amount of time between each administration of eachdose of vaccine composition is independently selected from the groupconsisting of 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months,6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months,13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, and 24 months.

In some embodiments of the invention, the first and second dengue virusvaccine compositions are the same. In alternative embodiments, the firstand second dengue virus vaccine compositions are not the same.

The dengue virus vaccine compositions of the invention can beadministered by different routes. In preferred embodiments of theinvention, the compositions of the invention are administeredparenterally, i.e. by intradermal, subcutaneous or intramuscularinjection. Subcutaneous and intramuscular administration can beperformed using, for example, needles or jet-injectors.

The compositions described herein may be administered in a mannercompatible with the dosage formulation, and in such amount as isimmunologically-effective to treat and/or reduce the likelihood ofdengue infection. The dose administered to a patient, in the context ofthe present invention, should be sufficient to affect a beneficialresponse in a patient over time such as a reduction in the level ofdengue virus, or to reduce the likelihood of infection by dengue. Thequantity of the dengue virus vaccines to be administered may depend onthe subject to be treated inclusive of the age, sex, weight and generalhealth condition thereof. In this regard, precise amounts of the vaccinerequired to be administered will depend on the judgment of thepractitioner. In determining the effective amount of the vaccine to beadministered in the treatment or prophylaxis against dengue infection,the physician may evaluate circulating plasma levels, progression ofdisease, and the production of anti-dengue antibodies. In any event,suitable dosages of the immunogenic compositions of the invention may bereadily determined by those of skill in the art.

Suitable dosing regimens are preferably determined taking into accountfactors well known in the art including age, weight, sex and medicalcondition of the patient; the route of administration; the desiredeffect; and the particular composition employed. The timing of dosesdepends upon factors well known in the art, and can range from 2 weeksto 24 months. After the initial administration one or more additionaldoses may be administered to maintain and/or boost antibody titers.

The invention also relates to methods for preventing dengue infection,or preventing or ameliorating the symptoms thereof, comprising the stepsof: administering to a patient in which dengue infection or the symptomsthereof are to be prevented or ameliorated compositions of the denguevirus vaccine. Further embodiments of this aspect of the inventioncomprise allowing a predetermined amount of time to pass afteradministration of the dengue virus vaccine composition, andadministering a second dose of a dengue virus vaccine composition.

In the method described above the first dengue vaccine is preferablytetravalent and comprises a DEN1, DEN2, DEN3, and DEN 4 component,wherein each component comprises either a live attenuated dengue virusor a live attenuated chimeric flavivirus, as described herein. Inexemplary embodiments, the live attenuated dengue vaccine comprises fourchimeric flaviviruses; wherein each of the chimeric flavivirus comprisesthe prM and E proteins of a single dengue virus serotype and the capsidand non-structural proteins of a different flavivirus, wherein the eachof the chimeric flavivirus is attenuated. In certain embodiments, thecapsid and nonstructural proteins of the four chimeric flaviviruses arefrom yellow fever virus. In alternative embodiments, the capsid andnonstructural proteins of each of the four chimeric flaviviruses arefrom a different dengue serotype than the prM and E proteins.

In some embodiments of this aspect of the invention, the second denguevaccine is a tetravalent recombinant dengue subunit vaccine comprisingdengue E proteins, or fragments thereof, from DEN1, DEN2, DEN3, andDEN4. Subunit vaccines useful in this method of the invention aredescribed herein. In preferred embodiments, the E proteins eachconstitute about 80% of the length of wild type E of DEN1, DEN2, DEN3and DEN4, starting from amino acid residue 1 at its N-terminus.

EXAMPLES

Examples of live attenuated dengue virus sequences used in these studiesare rDEN1—rDEN1Δ30-1545 PMVS (SEQ ID NO: 6); rDEN2—rDEN2/4 Δ30(ME)-1495,7163 PMVS (SEQ ID NO: 7); rDEN3—rDEN3Δ30/31-7164 PMVS (SEQID NO: 8); and rDEN4—rDEN4 Δ 30-7132,7163,8308 PMVS (SEQ ID NO: 9).

TABLE 1 Summary of PMVS DENV1 sequence changes Protein Amino NucleotideNucleotide Change Acid Amino Acid Change Number Gene wt PMVS Number wtPMVS 1544* E A C 484 Lys Arg 1545  E A G 484 Lys Arg 1549* E A G 485 SerSer *Introduced for stabilization and cloning purposes

TABLE 2 Summary of PMVS DENV2 sequence changes Nucleotide Change ProteinAmino Acid Change Original Amino Original Nucleotide cDNA Acid cDNANumber Gene Clone PMVS Number Clone PMVS 183 C T C 28 Leu Leu 1490 E G A184 Glu Glu 1495 E C U 186 Ser Phe 7132 NS4b C U 102 Thr Ile 7163 NS4b AC 112 Leu Phe 7166 NS4b C G 113 Val Val 7169 NS4b T C 114 His His

TABLE 3 Summary of PMVS DENV3 sequence changes Protein Amino NucleotideNucleotide Change Acid Amino Acid Change Number Gene wt PMVS Number wtPMVS 1539 E A G 202 Lys Arg 1681 E A G 250 Val Val 2095 E C U 388 Ile He7164 NS4b T C 115 Val Ala 7304 NS4b T C 162 Ser Pro 8082 NS5 A G 173 LysArg 10533 3′UTR G A N/A N/A N/A

TABLE 4 Summary of PMVS DENV4 sequence changes Nucleotide Change ProteinAmino Acid Change Original Amino Original Nucleotide cDNA Acid cDNANumber Gene Clone PMVS Number Clone PMVS 2440 NS1 T C 6 Val Ala 7132NS4b C U 102 Thr He 7153 NS4b T > C U 109 Val > Ala Val 7163 NS4b A C112 Leu Phe 8308 NS5 A > G G 249 Lys > Arg Arg

DENV1, 2, 3 and 4 wild type and original cDNA clone in the above tablescorrespond to the dengue virus serotype described in Whitehead, S. S. etal., J Virol 77:1653-1657 (2003); Blaney, J. E. et al. The Americanjournal of tropical medicine and hygiene 71:811-821 (2004); Blaney, J.E., Jr. et al., BMC Infect Dis 4:39 (2004); Durbin, A. P. et al., TheAmerican journal of tropical medicine and hygiene 65:405-413 (2001).

The above versions of the live attenuated dengue virus are referred toas DENV1 or DEN1, DENV2 or DEN2, DENV3 or DEN3 and DENV4 or DEN4 belowin the examples. For examples 1-6, the formulations had a potency of2×10⁵ pfu/ml of each of DENV1, DENV2, DENV3 or DENV4. For examples 7-10,the formulations had a potency of 1.5×10⁵ pfu/ml of each of DEN1, DEN2,DEN3 or DEN4.

Example 1

Effect of CMC, PG, and Amino Acids (Compared with Dengvaxia®Formulation) on DENV4

Three separate studies were performed to investigate the effects ofvarious excipients on the lyophilization yield and stability of DENV4.The formulations are listed in Table 5.

TABLE 5 Formulation Compositions Formulation Number Composition 1 11 mMpotassium phosphate, 90 mg/mL sucrose, 30 mM sodium chloride pH 7.5 2 11mM potassium phosphate, 90 mg/mL sucrose pH 7.5 3 11 mM potassiumphosphate, 90 mg/mL sucrose, 75 mM sodium chloride pH 7.5 4 11 mMpotassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose pH 7.5 5 11 mM potassium phosphate, 90mg/mL sucrose, 75 mM sodium chloride, 5 mg/mL sodiumcarboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 13 11 mMpotassium phosphate, 90 mg/mL sucrose, 25 mg/mL sorbitol, 75 mM sodiumchloride, 5 mg/mL sodium carboxymethylcellulose pH 7.5 18 11 mMpotassium phosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM Larginine pH 7.5 19 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mMsodium chloride, 5 mg/mL sodium carboxymethylcellulose, 5 mg/mLpropylene glycol, 25 mM L glutamic acid pH 7.5 20 11 mM potassiumphosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5 mg/mL sodiumcarboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM L leucine pH 7.521 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5mg/mL sodium carboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM Lproline pH 7.5 22 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mMsodium chloride, 5 mg/mL sodium carboxymethylcellulose, 5 mg/mL glycerolpH 7.5 25 11 mM TRIS, 90 mg/mL sucrose, 75 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 26 6 mMTRIS, 37.5 mg/mL sorbitol, 75 mg/mL sucrose, 55 mg/mL trehalose, 2.5mg/mL urea, 15 mg/mL amino acid mixture ^(‡) 45 11 mM potassiumphosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5 mg/mL sodiumcarboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 46 11 mMpotassium phosphate, 90 mg/mL sucrose, 30 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 47 11 mMpotassium phosphate, 90 mg/mL sucrose, 15 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 50 11 mMpotassium phosphate, 90 mg/mL sucrose, 50 mM potassium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol pH 7.5 55 11 mMpotassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL glycerol, 5 mg/mL urea pH 7.5 5611 mM potassium phosphate, 90 mg/mL sucrose, 201 mg/mL Leibovitz's L-15Medium without phenol red*, 5 mg/mL sodium carboxymethylcellulose, 5mg/mL propylene glycol pH 7.5 57 5.5 mM TRIS, 5.5 mM L histidine, 90mg/mL sucrose, 50 mM sodium chloride, 5 mg/mL sodiumcarboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM L leucine pH 7.581 11 mM potassium phosphate, 90 mg/mL sucrose, 75 mM sodium chloride, 5mg/mL sodium carboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM Lleucine pH 7.5 98 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mMsodium chloride, 5 mg/mL sodium carboxymethylcellulose, 5 mg/mLpropylene glycol, 25 mM L leucine, 0.01% poloxamer 188 pH 7.5 104 11 mMpotassium phosphate, 90 mg/mL sucrose, 50 mM sodium chloride, 5 mg/mLsodium carboxymethylcellulose, 5 mg/mL propylene glycol, 25 mM Lleucine, 25 mM L glutamic acid, pH 7.5 *Leibovitz's L-15 medium withoutphenol red is a solution manufactured by Hyclone Laboratories, Inc.

Study 1: DENV4 was formulated in 11 mM potassium phosphate, 90 mg/mLsucrose, and 75 mM NaCl (formulation 3), with the addition of 5 mg/mLsodium carboxymethylcellulose (sodium CMC) (formulation 4) or additionof 5 mg/mL Sodium CMC and 5 mg/mL propylene glycol (formulation 5).

Study 2: Formulation 5 was tested against comparable formulationscontaining either 25 mM leucine (formulation 20) or 25 mM proline(formulation 21) as well as 11 mM potassium phosphate, 90 mg/mL sucrose,50 mM NaCl, 5 mg/mL sodium CMC and 5 mg/mL propylene glycol.

Study 3: Formulation 20 was tested against a comparable formulationcontaining 11 mM potassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5mg/mL sodium CMC and 5 mg/mL propylene glycol and 25 mM glutamic acid(formulation 19) and the Dengvaxia® formulation (formulation 26), whichconsists of 37.5 mg/mL sorbitol, 75 mg/mL sucrose, 55 mg/mL trehalose,25 mg/mL urea, 6 mM TRIS, 15 mg/mL of an amino acid mixture.

For all studies, samples were frozen and a portion were stored at −70°C. as frozen liquid controls and a portion were lyophilized. Afterlyophilization, some samples were stored at −70° C. as control and theremainder were placed at 25° C. for 1 week. After incubation, the 25° C.samples were frozen and tested with a dengue relative infectivity assay(DRIA) along with the frozen liquid controls and frozen lyophilizedcontrols. Two individual vials of each sample were tested.

DRIA is a cell-based relative infectivity assay used to measureinfectivity of dengue virus formulation samples based on expression ofenvelope protein. Vero cells were plated in 96-well micro-titer plates,incubated for 24 hours, and then infected with serial dilutions of DEN1,DEN2, DEN3 and/or DEN4 reference standard and positive control specificfor the serotype being tested in addition to the test articles. Theinfected cells were incubated for 48 hours and followed by fixation ofthe cells with a dilute formaldehyde solution. The fixed cells were thenpermeabilized before primary antibody (rabbit anti-DEN serotype-specificMAb) was added to the plates and incubated overnight. After washing theplates, secondary antibody (Donkey NL637-conjugated anti-rabbit IgG, R&DSystems) was added to the wells and incubated at room temperature for >2hrs. After washing the plates, PBS was added to the wells in preparationfor image analysis using the MiniMax imaging reader (Molecular Devices).The relative potency (% RP) of samples (relative to the referencestandard) was calculated using SoftMAX Pro software (Molecular Devices)using a reduced 4 parameter logistic curve fit.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

A synergistic effect was observed with the combination of sodium CMC andpropylene glycol, which resulted in improved lyophilization yield andstability. The addition of leucine further improved yield and stability.Formulations containing Sodium CMC, propylene glycol and leucine orglutamic acid provided improved lyophilization yield over the Dengvaxiaformulation. See FIGS. 1-2. The 1 week 25° C. stability time point forformulation 26 was not tested due to cake collapse after storage at 25°C.

Example 2 Effect of Sugar Alcohol on DENV4:

DENV4 was formulated in a base formulation of 11 mM potassium phosphate,90 mg/mL sucrose, 75 mM NaCl, and 5 mg/mL sodium CMC pH 7.5 with 5 mg/mLpropylene glycol (formulation 5), 5 mg/mL glycerol (formulation 22), or25 mg/mL sorbitol (formulation 13) as sugar alcohols.

Samples were frozen and a portion were stored at −70° C. as frozenliquid controls and a portion were lyophilized. After lyophilization,some samples were stored at −70° C. and the remainder were placed at 25°C. for 1 week. After incubation, the 25° C. samples were frozen andtested with a dengue relative infectivity assay along with the frozenliquid controls and frozen lyophilized controls. Two individual vials ofeach sample were tested.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

This example demonstrates that both propylene glycol and glycerolimproved DENV4 lyophilization yield and stability compared to sorbitol(see FIGS. 3 and 4).

Example 3 Effect of pH on DENV4:

DENV4 was formulated in formulation 22 (11 mM potassium phosphate, 90mg/mL sucrose, 75 mM NaCl, 5 mg/mL sodium CMC, 5 mg/mL glycerol at pH7.0, 7.5 or 8.0).

Samples were frozen and a portion were stored at −70° C. as frozenliquid controls and a portion were lyophilized. After lyophilization,some samples were stored at −70° C. and the remainder were placed at 25°C. for 1 week. After incubation, the 25° C. samples were frozen andtested with a dengue relative infectivity assay along with the frozenliquid controls and frozen lyophilized controls. Two individual vials ofeach sample were tested.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

FIGS. 5 and 6 demonstrate that DENV4 can be formulated in formulation 22from pH 7.0 to pH 8.0.

Example 4 Effect of Buffer on DENV4:

In Study 1, DENV4 was formulated in a base formulation of 90 mg/mLsucrose, 75 mM NaCl, 5 mg/mL sodium CMC, and 5 mg/mL glycerol withalternative buffer systems adjusted to pH 7.5. Formulation 22 contained11 mM potassium phosphate and formulation 25 contained 11 mM TRIS inaddition to the base formulation.

In study 2, DENV4 was formulated in a base formulation of 90 mg/mLsucrose, 75 mM NaCl, 5 mg/mL sodium CMC, and 5 mg/mL propylene glycolwith alternative buffer systems adjusted to pH 7.5. Formulation 5contained 11 mM potassium phosphate and formulation 57 contained acombination of 5.5 mM histidine and 5.5 mM TRIS in addition to the baseformulation.

Samples were frozen and a portion were stored at −70° C. as frozenliquid controls and a portion were lyophilized. After lyophilization,some samples were stored at −70° C. and the remainder were placed at 25°C. for 1 week. After incubation, the 25° C. samples were frozen andtested with a dengue relative infectivity assay along with the frozenliquid controls and frozen lyophilized controls. Two individual vials ofeach sample were tested.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

FIGS. 7 and 8 demonstrate that DENV4 can be formulated in a variety ofbuffer systems at pH 7.5 including potassium phosphate, TRIS, or acombination of histidine and TRIS.

Example 5 Effect of NaCl on DENV4:

DENV4 was formulated in a base formulation of 11 mM potassium phosphate,90 mg/mL sucrose, 5 mg/mL sodium CMC, and 5 mg/mL propylene glycol witha concentration range of NaCl from 15-75 mM.

Samples were frozen and a portion were stored at −70° C. as frozenliquid controls and a portion were lyophilized. After lyophilization,some samples were stored at −70° C. and the remainder were placed at 25°C. for 1 week. After incubation, the 25° C. samples were frozen andtested with a dengue relative infectivity assay along with the frozenliquid controls and frozen lyophilized controls. Two individual vials ofeach sample were tested.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

FIGS. 9 and 10 show that DENV4 lyophilization yield and stability for 1week at 25° C. were similar when the NaCl concentration ranged from15-75 mM.

Example 6 Effect of Propylene Glycol and Glycerol on all DengueSerotypes:

DENV1, DENV2, DENV3 and DENV4 were prepared as monovalent drug productsin formulation 5 (11 mM potassium phosphate, 90 mg/mL sucrose, 75 mMNaCl, 5 mg/mL CMC, 5 mg/mL propylene glycol), formulation 20 (11 mMpotassium phosphate, 90 mg/mL sucrose, 50 mM NaCl, 5 mg/mL CMC, 5 mg/mLpropylene glycol, and 25 mM Leucine) and formulation 22 (11 mM potassiumphosphate, 90 mg/mL sucrose, 75 mM NaCl, 5 mg/mL CMC, 5 mg/mL glycerol)at pH 7.5.

Samples were frozen and a portion were stored at −70° C. as frozenliquid controls and a portion were lyophilized. After lyophilization,some samples were stored at −70° C. and the remainder were placed at 25°C. for 1 week. After incubation, the 25° C. samples were frozen andtested with a dengue relative infectivity assay along with the frozenliquid controls and frozen lyophilized controls. Two individual vials ofeach sample were tested.

Lyophilization yields were calculated by dividing the lyophilizedinfectivity result by the frozen liquid control infectivity result. Tocalculate log loss after storage at 25° C. for one week, infectivityvalues were converted into log scale and the 1 week 25° C. log resultwas subtracted from the −70° C. lyophilized control result for eachformulation.

FIGS. 11 and 12 show that propylene glycol and glycerol stabilize allfour serotypes in combination with sucrose, NaCl, and Sodium CMC.

Example 7

SPG (Sucrose, Potassium Phosphate, Glutamic acid) was made as a 10×solution with the below concentration seen in the Table 6.

TABLE 6 Sucrose (crystals) 746.2 mg/mL KH₂PO₄ (monobasic, anhydrous) 5.17 mg/mL K₂HPO₄ (dibasic, anhydrous) 12.54 mg/mL L-glutamic acid(monosodium salt, monohydrate)  11.2 mg/mL

The following other solutions were also made: 650 mg/mL Sucrose, 650mg/mL Trehalose, dihydrate, 200 mg/mL Gelatin, 150 mg/mL Arginine, and20 mg/mL Human Serum Albumin (HSA).

The SPG, sucrose, trehalose, gelatin, arginine, and HSA were filteredwith PES 0.22 μm Stericup filters. The solutions, Leibovitz's L-15 andDEN1 were combined to obtain the final formulations seen below:

TABLE 7 Rx# Formulations 1 250 mg/mL L-15, 11 mM Potassium Phosphate, 6mM L-glutamic acid, 75 mg/mL Sucrose 2 250 mg/mL L-15, 11 mM PotassiumPhosphate, 6 mM L-glutamic acid, 75 mg/mL Sucrose, 175 mg/mL Trehalose 4250 mg/mL L-15, 11 mM Potassium Phosphate, 6 mM L-glutamic acid, 75mg/mL Sucrose , 175 mg/mL Trehalose, 2.5 mg/mL HAS 5 250 mg/mL L-15,11mM Potassium Phosphate, 6 mM L-glutamic acid, 75 mg/mL Sucrose , 175mg/mL Trehalose, 25 mg/mL Gelatin 6 250 mg/mL L-15, 11 mM PotassiumPhosphate, 6 mM L-glutamic acid, 150 mg/mL Sucrose 7 250 mg/mL L-15, 11mM Potassium Phosphate, 6 mM L-glutamic acid, 75 mg/mL Sucrose, 75 mg/mLTrehalose 9 250 mg/mL L-15, 11 mM Potassium Phosphate, 6 mM L-glutamicacid, 75 mg/mL Sucrose, 75 mg/mL Trehalose, 40 mg/mL Arginine 11 450mg/mL L-15, 11 mM Potassium Phosphate, 6 mM L-glutamic acid, 75 mg/mLSucrose 12 450 mg/mL L-15, 11 mM Potassium Phosphate, 6 mM L-glutamicacid, 75 mg/mLSucrose, 175 mg/mL Trehalose, 25 mg/mL Gelatin 13 450mg/mL L-15, 11 mM Potassium Phosphate, 6 mM L-glutamic acid, , 75 mg/mLSucrose, 75 mg/mL Trehalose, 40 mg/mL Arginine

The formulations were filled into 2R glass vials at a 0.5 mL fill andfrozen at −115° C. for 15 minutes. Once frozen the vials were dried inthe Microwave Vacuum Dryer (MVD). Once dried, some vials were place onstability at 25° C. for 1 week. The vials were then submitted forpotency testing using the Dengue Relative Infectivity Assay (DRIA).

Microwave Drying Process

Material being dried in the microwave vacuum dryer (MVD) were blastfrozen and immediately loaded into the drying chamber. Vacuum wasquickly pulled on the chamber to below 100 mTorr. Once the vacuumsetpoint was reached, the magnetrons (i.e. number of magnetron and poweroutput) were selected to begin drying the material. Microwave (radiationis applied in a travelling wave format) was operated in scanning modewith an algorithm that cycles selected magnetrons (e.g. 2 magnetrons outof 4 total magnetrons) on and off every 30 seconds for uniform powerdistribution. Furthermore, a water load on top of the unit allows forsingle pass of microwave through the sample that minimizes anyinterference from reflected microwave thereby allowing for controlledsublimation. Power was increased throughout the drying cycle to achievea final terminal temperature of 30-45° C. Once dried, vacuum was brokenin the chamber and the material was sealed.

TABLE 8 4 5 2 250 mg/mL L-15, 250 mg/mL L-15, 7 1 250 mg/mL L-15, 11 mMPotassium 11 mM Potassium 6 250 mg/mL L-15, 250 mg/mL L-15, 11 mMPotassium Phosphate, 6 mM Phosphate, 6 mM 250 mg/mL L-15, 11 mMPotassium 11 mM Potassium Phosphate, 6 mM L-glutamic acid, L-glutamicacid, 11 mM Potassium Phosphate, 6 mM Phosphate, 6 mM L-glutamic acid,75 mg/mL Sucrose, 75 mg/mL Sucrose, Phosphate, 6 mM L-glutamic acid, Rx#L-glutamic acid, 75 mg/mL Sucrose, 175 mg/mL Trehalose, 175 mg/mLTrehalose, L-glutamic acid, 75 mg/mL Sucrose, Rx 75 mg/mL Sucrose 175mg/mL Trehalose 2.5 mg/mL HSA 25 mg/mL Gelatin 150 mg/mL Sucrose 75mg/mL Trehalose F/T 55 88 142 102 61 69 Yield (%) Drying 62 59 67 83 6478 Yield (%) Avg. 0.45 0.26 0.23 0.19 0.33 0.35 Log Loss 9 12 13 250mg/mL L-15, 450 mg/mL L-15, 450 mg/mL L-15, 11 mM Potassium 11 11 mMPotassium 11 mM Potassium Phosphate, 6 mM 450 mg/mL L-15, Phosphate, 6mM Phosphate, 6 mM L-glutamic acid, 11 mM Potassium L-glutamic acid,L-glutamic acid, , 75 mg/mL Sucrose, Phosphate, 6 mM 75 mg/mL Sucrose,75 mg/mL Sucrose, Rx# 75 mg/mL Trehalose, L-glutamic acid, 175 mg/mLTrehalose, 75 mg/mL Trehalose, Rx 40 mg/mL Arginine 75 mg/mL Sucrose 25mg/mL Gelatin 40 mg/mL Arginine F/T 53 23 98 29 Yield (%) Drying 6 16798 10 Yield (%) Avg. 3.65 0.36 0.27 2.11 Log Loss

Freeze/Thaw (F/T) yield, drying yield, and log loss at 25° C. for 1 weekwere determined for all formulations. Freeze/thaw (F/T) yield wascalculated by dividing the reported relative potency by the expectedrelative potency for the frozen controls at −70° C. Drying yield wascalculated by dividing the relative potency of the dried material by therelative potency of the frozen control. The log loss was calculated byconverting the relative potency of the TO timepoint of the driedmaterial and the 1 week 25° C. stability material into logs by a Log 10calculation. Once the numbers are converted into log, the stabilitytimepoint was subtracted from the TO timepoint to determine the log lossat 25° C. for 1 week.

Formulations 2, 4, 5, and 12 showed the best combination of F/T yield,drying yield, and log loss at 25° C. for one week. All four of theseformulations contained ≥25% disaccharide (sucrose and/or trehalose).

TABLE 9 summary of ranges of excipients of formulations in Table 7Excipients Quantity (per 0.5 mL dose) Sucrose 37.5 mg-75 mg   PotassiumPhosphate (monobasic, ~0.26 mg anhydrous) Potassium Phosphate (dibasic,anhydrous) ~0.63 mg L-glutamic acid (monosodium salt, 0.56 mgmonohydrate) Trehalose 37.5 mg-87.5 mg Human Serum Albumin (HSA) 1.25 mgArginine 20 mg Gelatin 12.5 mg

Example 8

SPG (Sucrose, Potassium Phosphate, Glutamic acid) was made according toExample 7. The following other solutions were also made: 650 mg/mLSucrose, 650 mg/mL Trehalose, 5M Sodium Chloride (NaCl), and 10 mg/mLsodium Carboxymethyl Cellulose (sodium CMC).

All solutions were filter with PES 0.22 μm Stericup filters. Thesolutions and Dengue virus DEN1 or DEN4 were combined to obtain thefinal formulations seen in the results table.

The formulations were filled into 2R glass vials at a 0.5 mL fill andfrozen at −115° C. for 15 minutes. Once frozen the vials were dried inthe Microwave Vacuum Dryer (MVD). Once dried, some vials were place onstability at 25° C. for 1 week. The vials were then submitted forpotency testing using the Dengue Relative Infectivity Assay (DRIA).

TABLE 10 DEN1 DEN4 Avg. Log Loss Avg. Log Loss Residual Formulations 25°C. 1 week 25° C. 1 week Moisture (%) 11 mM Potassium Phosphate, 6 mML-glutamic 0.55 0.55 5.65 ± 0.75 acid, 75 mg/mL Sucrose, 175 mg/mLTrehalose 11 mM Potassium Phosphate, 6 mM L-glutamic 0.46 0.54 5.61 ±0.40 acid, 75 mg/mL Sucrose, 175 mg/mL Trehalose, 30 mM NaCl 11 mMPotassium Phosphate, 6 mM L-glutamic 0.60 0.59 4.58 ± 0.33 acid, 75mg/mL Sucrose, 175 mg/mL Trehalose, 30 mM NaCl, 5 mg/mL sodium CMC 11 mMPotassium Phosphate, 6 mM L-glutamic 1.12 0.95 3.71 ± 0.22 acid, 75mg/mL Sucrose, 75 mg/mL Trehalose 11 mM Potassium Phosphate, 6 mML-glutamic 0.96 0.51 4.75 ± 0.54 acid, 75 mg/mL Sucrose, 75 mg/mLTrehalose, 30 mM NaCl 11 mM Potassium Phosphate, 6 mM L-glutamic 1.070.65 3.98 ± 0.01 acid, 75 mg/mL Sucrose, 75 mg/mL Trehalose, 30 mM NaCl,5 mg/mL sodium CMC 11 mM Potassium Phosphate, 6 mM L-glutamic 0.83 1.822.20 ± 0.12 acid, 75 mg/mL Trehalose 11 mM Potassium Phosphate, 6 mML-glutamic 1.18 1.33 3.43 ± 0.19 acid, 75 mg/mL Trehalose, 30 mM NaCl

The log loss was calculated by converting the relative potency of the TOtimepoint of the dried material and the 1 week 25° C. stability materialinto logs by a Log 10 calculation. Once the numbers were converted intolog, the stability timepoint was subtracted from the TO timepoint todetermine the log loss at 25° C. for 1 week.

The results showed that for DEN1 formulations containing ≥25%disaccharides, the lowest log loss at 25° C. one week was observed. TheDEN4 formulations with ≥15% dissaccharides and 30 mM salt had the lowestlog loss at 25° C. at one week. For formulations containing ≥15%dissaccharides and salt, the addition of sodium CMC helped with reducingresidual moisture.

TABLE 11 summary of ranges of excipients of formulations in Table 10Excipients Quantity (per 0.5 mL dose) Sucrose   0 mg-37.5 mg PotassiumPhosphate (monobasic, ~0.26 mg anhydrous) Potassium Phosphate (dibasic,anhydrous) ~0.63 mg L-glutamic acid (monosodium salt, 0.56 mgmonohydrate) Trehalose 37.5 mg-87.5 mg Sodium Carboxylmethyl cellulose2.5 mg Sodium Chloride 0.88 mg

Example 9

Formulations were 1×SPG (11 mM Potassium Phosphate, 6 mM L-glutamicacid, 0.22M Sucrose) with varying amounts of L-15 (90%, 45%, and 25%)and DEN1, DEN2, DEN3, or DEN4. The major component in Leibovitz's L-15is NaCl (137.39 mM). Therefore, 90% L-15 equals 123.65 mM NaCl, 45% L-15equals 61.83 mM NaCl, and 25% L-15 equals 34.35 mM NaCl.

The formulations were filled into 2R glass vials at a 0.5 mL fill andfrozen at −115° C. for 15 minutes. Once frozen the vials were dried inthe Microwave Vacuum Dryer (MVD). The vials were then submitted forpotency testing using the Dengue Relative Infectivity Assay (DRIA).

Formulations that were dried fast under MVD were more stable thanformulations dried slower under lyophilization (See FIGS. 13-16). In theabove formulation, salt concentration of ≥61.83 mM NaCl appears toimprove DEN4 stability during drying that is not observed in the otherthree types (DEN1, DEN2, and DEN3).

Example 10

The following solutions were also made: 500 mg/mL Sucrose, 250 mg/mLTrehalose, 1M Sodium Chloride (NaCl), and 10 mg/mL Sodium CarboxymethylCellulose (Sodium CMC), 100 mg/mL Polyvinylpyrrolidone (PVP), 100 mMPotassium Phosphate, 500 mg/mL Propylene Glycol, and Sterile Water.

All solutions were filtered with PES 0.22 μm Stericup filters. Thesolutions and Dengue virus (DEN1) were combined to obtain the followingformulations: 11 mM Potassium Phosphate, 90 mg/mL Sucrose, 5 mg/mLsodium CMC, 5 mg/ml PG; 11 mM Potassium Phosphate, 200 mg/mL Sucrose, 50mg/ml PVP K12; 11 mM Potassium Phosphate, 75 mg/mL Sucrose, 175 mg/mlTrehalose, 30 mM NaCl, 5 mg/ml CMC.

The formulations were filled into 2R glass vials at a 0.5 mL fill andfrozen at −115° C. for 15 minutes. Once frozen the vials were dried ineither a Microwave Vacuum Dryer (MVD) or Lyophilizer (Lyo). The vialswere then submitted for potency testing using the Dengue RelativeInfectivity Assay (DRIA).

FIG. 17 shows that relative potency of vials for the formulations driedin the Microwave Vacuum Dryer is greater than or equal to those dried inthe Lyophilizer.

TABLE 12 summary of ranges of excipients of formulations in FIG. 17Excipients Quantity (per 0.5 mL dose) Sucrose 37.5 mg-100 mg PotassiumPhosphate (monobasic, ~0.26 mg anhydrous) Potassium Phosphate (dibasic,anhydrous) ~0.63 mg Trehalose 87.5 mg Sodium carboxymethyl cellulose 2.5mg Sodium Chloride 0.88 mg PVP K12 25 mg Propylene Glycol 2.5 mg

Example 11

Tetravalent formulations (formulation 20) of DENV1, DENV2, DENV3 andDENV4 were lyophilized and stored at 37° C. for one week (FIG. 18A), 25°C. for one month (FIG. 18B), and 2-8° C. for 18 months (FIGS. 19A-D).Potency was analyzed by plaque assay (as described earlier in the text)at each time point. A control sample stored at −70° C. was tested byplaque assay in the same assay run as each stability time point. A logloss for each time point was calculated by subtracting the log result ofthe stability sample from the −70° C. control sample. FIGS. 18A-B and19A-D show the log loss over time for each of the serotypes in thetetravalent formulation 20. The error bars indicate two standard errorof the mean of the log loss calculated at each time point. Formulation20 provides thermal stability to all four dengue serotypes in thetetravalent vaccine at 37° C., 25° C. and 2-8° C. as evidenced by theminimal potency loss observed at 1 week, 1 month and 18 months,respectively.

Example 12 High Throughput Plaque Assay

The high throughput plaque assay “microplaque (g)” assay is anautomated, miniaturized dengue plaque assay run in a 96-well microplate.Briefly, Vero cells are seeded into black-walled, clear bottomtissue-culture plates in OptiPro SFM with 2% L-glutamine at 40,000 cellsper well. Cells are allowed to attach overnight at 37° C., 5% pCO₂, >90%rH. Virus is pre-diluted in OptiMEM reduced serum media and furtherserially diluted 1:2 in media in ultra-low attachment plates. The plantmedium is removed from the cell plates using gentle aspiration, and 25μL/well of incolum is transferred from the serial dilution plate to thecell plate. Viral adsorption proceeds for 4 hours at 37° C., 5%pCO₂, >90% rH. After the adsorption incubation, 175 μL/well overlaymedium is added to all wells to inhibit viral secretion and spread.Depending on serotype, infection proceeds for 2 or 3 days at theaforementioned incubation conditions.

After the infection incubation, overlay medium is removed and cells arefixed with 3.7% formaldehyde in PBS. Plates are permeabilized with 0.5%Triton X-100 in PBS, then blocked with 1% BSA in PBS. Type specificrabbit monoclonal antibodies, followed by anti-rabbit AlexaFluor488 areused to fluorescently stain viral plaques. Plates are imaged using aPerkin Elmer EnSight and fluorescent plaques are counted by an automatedcounting algorithm. Titer is determiend using equation below from wellsthat contain valid object counts that are within counting criteria (typedependent):

${{Viral}\mspace{14mu} {{Titer}( \frac{PFU}{mL} )}} = {\frac{{plaques}\mspace{14mu} {counted}}{{volume}\mspace{14mu} {of}\mspace{14mu} {inoculum}\mspace{14mu} ({mL})} \times {total}\mspace{14mu} {dilution}}$

Two studies were executed in which tetravalent formulations of DENV1,DENV2, DENV3 and DENV4 were lyophilized in formulations detailed inTable 13 and stored at 25° C. for one week. Each formulation contains 9%sucrose, 11 mM potassium phosphate, 50 mM NaCl, 25 mM Leu at pH 7.5 andvarying amounts of CMC or PG. Potency was analyzed by the highthroughput plaque assay described above at each time point. A controlsample stored at −70° C. was also tested in the assay. A log loss foreach time point was calculated by subtracting the log result of thestability sample from the −70° C. control sample. Tables 14a and 14bshow the log loss over time for each of the serotypes in the varioustetravalent formulations. Concentrations of 0.2%-1% CMC or PG in variouscombinations show similar stability to each other and increasedstability over formulations without the combination.

TABLE 13 Tetravalent Formulations Formulation Full FormulationFormulation Variations Number 9% sucrose, 11 mM potassium phosphate, 50mM No CMC or PG 140 NaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mM potassiumphosphate, 0.5% CMC only 141 CMC, 50 mM NaCl, 25 mM Leu, pH 7.5 9%sucrose, 11 mM potassium phosphate, 0.2% 0.2% CMC, 0.2% PG 142 CMC, 0.2%propylene glycol, 50 mM NaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mMpotassium phosphate, 0.3% 0.3% CMC, 0.3% PG 143 CMC, 0.3% propyleneglycol, 50 mM NaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mM potassiumphosphate, 0.5% 0.5% CMC, 0.5% PG 20 CMC, 0.5% propylene glycol, 50 mMNaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mM potassium phosphate, 0.8% 0.8%CMC, 0.8% PG 144 CMC, 0.8% propylene glycol, 50 mM NaCl, 25 mM Leu, pH7.5 9% sucrose, 11 mM potassium phosphate, 0.9% 0.9% CMC, 0.9% PG 145CMC, 0.9% propylene glycol, 50 mM NaCl, 25 mM Leu, pH 7.5 9% sucrose, 11mM potassium phosphate, 0.8% 0.8% CMC, 0.5% PG 138 CMC, 0.5% propyleneglycol, 50 mM NaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mM potassiumphosphate, 0.5% 0.5% CMC, 0.8% PG 139 CMC, 0.8% propylene glycol, 50 mMNaCl, 25 mM Leu, pH 7.5 9% sucrose, 11 mM potassium phosphate, 0.3% 0.3%CMC, 0.5% PG 123 CMC, 0.5% propylene glycol, 50 mM NaCl, 25 mM Leu, pH7.5

TABLE 14a Effect of Concentration of CMC and PG on Stability at 25° C.Formulation DENV1 Log Loss DENV2 Log Loss DENV3 Log Loss 1 DENV4 LogLoss Formulation Number 1 week 25° C. 1 week 25° C. week 25° C. 1 week25° C. No CMC or PG 140 0.41 0.38 0.42 0.43 CMC only 141 0.47 0.40 0.520.31 0.2% CMC, 0.2% PG 142 0.23 0.20 0.27 0.10 0.3% CMC, 0.3% PG 1430.27 0.18 0.15 0.04 0.5% CMC, 0.5% PG 20 0.18 0.16 0.23 0.04 0.8% CMC,0.8% PG 144 0.22 0.17 0.09 0.22 0.9% CMC, 0.9% PG 145 0.16 0.12 0.030.16 0.8% CMC, 0.5% PG 138 0.15 0.07 0.21 −0.01 0.5% CMC, 0.8% PG 1390.20 0.12 0.18 0.04 0.3% CMC, 0.5% PG 123 0.22 0.13 0.10 0.08

TABLE 14b Effect of Concentration of CMC and PG on Stability at 25° C.Formulation DENV1 Log LOSS DENV2 Log Loss DENV3 Log Loss 1 DENV4 LogLOSS Formulation Number 1 week 25° C. 1 week 25° C. week 25° C. 1 week25° C. 0.5% CMC, 0.5% PG 20 0.19 0.30 0.15 0.06 0.2% CMC, 0.5% PG 1220.20 0.15 0.21 0.07 0.3% CMC, 0.5% PG 123 0.26 0.12 0.16 0.02 0.4% CMC,0.5% PG 124 0.26 0.33 0.33 0.10 0.1% CMC, 0.5% PG 125 0.27 0.33 0.220.16 0.5% CMC, 0.2% PG 126 0.23 0.20 0.30 0.11 0.5% CMC, 0.3% PG 1270.31 0.28 0.22 0.14 0.5% CMC, 0.7% PG 128 0.24 0.20 0.15 0.14 0.5% CMC,0.1% PG 129 0.23 0.15 0.16 0.19

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention.

Having described different embodiments of the invention herein withreference to the accompanying drawings, it is to be understood that theinvention is not limited to those precise embodiments, and that variouschanges and modifications may be effected therein by one skilled in theart without departing from the scope or spirit of the invention asdefined in the appended claims.

1. A formulation comprising a live attenuated dengue vaccine comprisingat least one live attenuated dengue virus (LAV) or at least one liveattenuated chimeric flavivirus (LACV), a buffer at pH about 6.5 to 8.5,a sugar, a glycol or sugar alcohol, and a cellulose derivative selectedfrom the group consisting of carboxymethyl cellulose, hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethylcellulose (2-HEC), crosscarmellose, and methyl cellulose, or apharmaceutically acceptable salt thereof, and optionally an alkali oralkaline salt and/or an amino acid selected from the group consisting ofAla, Asp, His, Leu, Lys, Gln, Pro, Glu, and a combination thereof. 2.The formulation of claim 1, wherein the buffer is selected from thegroup consisting of succinate, histidine, phosphate, TRIS, Bis-Tris,MES, MOPS, HEPES, acetate, citrate, and a combination thereof.
 3. Theformulation of claim 1, wherein the alkali or alkaline salt is magnesiumchloride, calcium chloride, potassium chloride, sodium chloride or acombination thereof.
 4. The formulation of claim 1, wherein the sugar istrehalose or sucrose.
 5. The formulation of claim 1, wherein thecellulose derivative is a pharmaceutically acceptable salt ofcarboxymethyl cellulose.
 6. The formulation of claim 1, wherein theglycol is selected from the group consisting of propylene glycol,polypropylene glycol, ethylene glycol, polyethylene glycol, andpolyethylene glycol monomethyl ethers.
 7. The formulation of claim 1,wherein the glycol or sugar alcohol is propylene glycol or glycerol. 8.The formulation of claim 1 that comprises a live attenuated denguevaccine comprising at least one live attenuated dengue virus (LAV) or atleast one live attenuated chimeric flavivirus at about 100-10,000,000pfu/ml, a buffer at pH about 6.5 to 8.5, about 50-300 mg/ml sugar, about2.5-10.0 mg/ml propylene glycol (PG) or glycerol, and about 0.3-10 mg/mlsodium carboxymethylcellulose (sodium CMC), and optionally about 10-150mM NaCl and/or about 10-100 mM amino acid selected from the groupconsisting of Ala, Asp, His, Leu, Lys, Gln, Pro, Glu and a combinationthereof.
 9. The formulation of claim 1 that comprises the liveattenuated dengue vaccine at about 100-100,000 pfu/ml, about 5-300 mMhistidine, TRIS, Bis-Tris or phosphate buffer, or a combination thereofat pH about 7.0 to 8.0, about 50-300 mg/ml sugar, about 3-10 mg/mlpropylene glycol or glycerol, and about 3-10 mg/ml sodiumcarboxymethylcellulose, and optionally about 15-75 mM NaCl and/or about10-75 mM amino acid selected from the group consisting of Ala, Asp, His,Leu, Lys, Gln, Pro, Glu and a combination thereof.
 10. The formulationof claim 1 that comprises the live attenuated dengue vaccine at about600-20,000 pfu/ml, about 5-300 mM potassium phosphate buffer at pH about7.0-8.0, about 60-120 mg/ml sucrose or trehalose or a combinationthereof, about 3-7 mg/ml propylene glycol or glycerol, and about 3-7mg/ml sodium carboxymethylcellulose with average molecular weight ofabout 90,000, and about 30-90 mM NaCl, and optionally about 10-75 mMamino acid Leu, Lys or Glu, or a combination thereof.
 11. Theformulation of claim 1 that comprises the live attenuated dengue vaccineat about 600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pHabout 7.0-8.0, about 90 mg/ml sucrose, about 5 mg/ml propylene glycol orglycerol, about 5 mg/ml sodium carboxymethylcellulose with averagemolecular weight of about 90,000, and about 75 mM NaCl.
 12. Theformulation of claim 1 that comprises the live attenuated dengue vaccineat about 600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pHabout 7.0-8.0, about 90 mg/ml sucrose, about 5 mg/ml propylene glycol,about 5 mg/ml sodium carboxymethylcellulose with average molecularweight of about 90,000, about 50 mM NaCl, and about 25 mM Leu.
 13. Theformulation of claim 1 that comprises the live attenuated dengue vaccineat about 600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pHabout 7.5, about 90 mg/ml sucrose, about 5 mg/ml propylene glycol, about5 mg/ml sodium carboxymethylcellulose with average molecular weight ofabout 90,000, and about 30 mM NaCl.
 14. The formulation of claim 9,further comprising about 90-200 mg/ml trehalose.
 15. The formulation ofclaim 1 that comprises the live attenuated dengue vaccine at about600-20,000 pfu/ml, about 11 mM potassium phosphate buffer at pH about7.5-8, about 90 mg/ml sucrose, about 110 mg/ml trehalose, about 5 mg/mlpropylene glycol, about 5 mg/ml sodium carboxymethylcellulose withaverage molecular weight of about 90,000, about 50 mM NaCl, and about 25mM Leu.
 16. The formulation of claim 1, wherein the formulation furthercomprises a surfactant selected from poloxamer 188 and poloxamer 407 atabout 0.0001 to 5% w/v.
 17. A formulation that comprises a liveattenuated dengue vaccine comprising at least one live attenuated denguevirus (LAV) or at least one live attenuated chimeric flavivirus at about20-200,000,00 pfu/ml, a buffer at pH about 6.5 to 8.5, a sugar at about150-300 mg/ml, a carrier selected from the group consisting ofpolyvinylpyrrolidone (PVP), carboxymethyl cellulose, hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HPMC), 2-hydroxyethylcellulose (2-HEC), crosscarmellose, and methyl cellulose or apharmaceutically acceptable salt thereof, Human Serum Albumin (HSA) andgelatin; and optionally an alkali salt or alkaline salt at about 5-100mM and/or an amino acid Gln, Pro or Glu, or a combination thereof. 18.The formulation of claim 17, wherein the buffer is selected from thegroup consisting of succinate, histidine, phosphate, TRIS, Bis-Tris,MES, MOPS, HEPES, acetate, citrate, and a combination thereof.
 19. Theformulation of claim 17, wherein the alkali or alkaline salt ismagnesium chloride, calcium chloride, potassium chloride, sodiumchloride or a combination thereof.
 20. The formulation of claim 17,wherein the sugar is trehalose or sucrose, or a combination thereof. 21.The formulation of claim 20, wherein the sucrose to trehalose ratio isbetween 1:1 to 1:4.
 22. The formulation of claim 17, wherein the carrieris a sodium carboxymethyl cellulose, HPMC, HSA or gelatin.
 23. Theformulation of claim 17 that comprises the live attenuated denguevaccine at about 100-10,000,000 pfu/ml, a buffer at pH about 6.5-8.0,about 150-300 mg/ml sugar as a combination of sucrose and trehalose,about 0.3 to 40 mg/ml sodium CMC, HSA, HPMC or gelatin, and optionallyabout 10-100 mM alkali or alkaline salt and/or about 5-25 mM glutamicacid.
 24. The formulation of claim 17 that comprises the live attenuateddengue vaccine at about 100-100,000 pfu/ml, about 5-300 mM histidine,TRIS or phosphate buffer, or a combination thereof at pH about 7.0 to8.0, about 50-100 mg/ml sucrose, about 90-200 mg/ml trehalose, about0.3-10 mg/ml sodium CMC or about 10-40 mg/ml gelatin, and about 30-90 mMalkali or alkaline salt.
 25. The formulation of claim 17 that comprisesthe live attenuated dengue vaccine at about 600-20,000 pfu/ml, about5-20 mM potassium phosphate at pH about 7.0-8.0, about 75 mg/ml sucrose,about 175 mg/ml trehalose, about 5 mg/ml sodium CMC with averagemolecular weight of about 90,000, and about 30 mM NaCl.
 26. Theformulation of claim 17 that comprises the live attenuated denguevaccine at about 600-20,000 pfu/ml, about 5-20 mM potassium phosphate atpH about 7.0-8.0, about 75 mg/ml sucrose, about 175 mg/ml trehalose,about 25 mg/ml gelatin, and about 30 mM NaCl.
 27. The formulation ofclaim 17 that comprises the live attenuated dengue vaccine at about600-20,000 pfu/ml, about 5-20 mM potassium phosphate at pH about 7-8,about 250 mg/ml sucrose, and about 50 mg/ml PVP K12.
 28. The formulationof claim 17, further comprising a surfactant selected from poloxamer 188and poloxamer 407 at about 0.0001 to 5% w/v.
 29. The formulation ofclaim 1 that further comprises an aluminum adjuvant.
 30. The formulationof claim 1 that is frozen or lyophilized.
 31. The formulation of claim 8that is reconstituted in solution.
 32. The formulation of claim 8 thatis an aqueous solution prior to lyophilization or microwave vacuumdrying.
 33. The formulation of claim 31, wherein the reconstitution isperformed with solution is about 0.5-1.0 ml saline solution, water orBacteriostatic Water for Injection (BWFI) and optionally comprises adiluent comprising an aluminum adjuvant.
 34. The formulation of claim 1,wherein the live attenuated dengue vaccine is tetravalent.
 35. Theformulation of claim 1, wherein the LAV or the LACV comprise a viralgenome that contains a deletion of about 30 nucleotides corresponding tothe TL-2 stem-loop structure of the 3′ untranslated (UTR) region (UTR).36. The formulation of claim 1, wherein the live attenuated dengue virus(LAV) comprises a viral genome that contains a deletion of about 30nucleotides corresponding to the TL-2 stem-loop structure of the 3′untranslated (UTR) region (UTR), and is immunogenic against dengueserotype 3, wherein the viral genome of the LAV further contains adeletion of nucleotides upstream from the Δ30 deletion corresponding tothe TL-3 structure of the 3′UTR.
 37. The formulation of claim 1, whereinthe live attenuated dengue virus (LAV) comprises rDEN1Δ30, rDEN2/4Δ30,rDEN3Δ30/31, and rDEN4Δ30.
 38. The formulation of claim 1, wherein thelive attenuated dengue virus (LAV) comprises rDEN1Δ30-1545, rDEN2/4Δ30(ME)-1495,7163, rDEN3Δ30/31-7164, and rDEN4Δ30-7132,7163,8308.